新規試験法提案書

皮膚感作性試験代替法
Interleukin-8 Reporter Gene Assay（IL-8 Luc assay）

令和 2 年12月

国立医薬品食品衛生研究所

新規試験法提案書
令和 2 年 12 月 2 日
No.

皮膚感作性試験代替法

2020-02

Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)
に関する提案

令和 2 年 6 月 25 日に国立医薬品食品衛生研究所にて開催された新規試験法評価会議（通称：JaCVAM 評価会議）において以下の提案がなされた。
提案内容： 本試験法において陽性の結果が得られた場合、皮膚感作性物質と判定することは可能であるが、感作性の強度を分類することは困難である。また、陰性の結果が得られた
場合、非皮膚感作性物質と判定できない。したがって本試験法単独での皮膚感作性の判定は不十分であり、被験物質の特性を十分に理解した上で、IATA（Integrated
Approaches to Testing and Assessment）を構成するその他の情報と組み合わせて適切に評価することが必要である。なお、本試験法の利用にあたっては、適用範囲および判
定不可物質の判断に十分に配慮した上で使用されるべきである。
この提案書は、皮膚感作性試験資料編纂委員会によりまとめられた文書を用いて、JaCVAM評
価会議が評価および検討した結果、その有用性が確認されたことから作成された。
以上の理由により、行政当局の安全性評価方法として皮膚感作性試験代替法 IL-8 Luc assay の使用を提案するものである。

大野泰雄
JaCVAM 評価会議

平林容子
議長

JaCVAM 運営委員会

委員長

i

JaCVAM 評価会議
大 野 泰 雄 （公益財団法人 木原記念横浜生命科学振興財団）：座長
五十嵐良明 （国立医薬品食品衛生研究所）
池 田 孝 則 （日本製薬工業協会）**
石 井 雄 二 （国立医薬品食品衛生研究所

安全性生物試験研究センター）

稲 若 邦 文 （日本化学工業協会）
井 上 智 彰 （日本免疫毒性学会）*
今 井 教 安 （日本動物実験代替法学会）
岩瀬裕美子 （日本製薬工業協会）*
久 保 文 宏 （独立行政法人 医薬品医療機器総合機構）*
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 川 秋 佳 （国立医薬品食品衛生研究所

病理部／済生会宇都宮病院）

西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
沼澤聡 （日本毒性学会）

平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
廣 田 衞 彦 （日本化粧品工業連合会）
増 村 健 一 （日本環境変異原学会）
山 本 恵 子 （独立行政法人 医薬品医療機器総合機構）**
横 関 博 雄 （日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 9 月 30 日*：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日**：令和 2 年 4 月 1 日～令和 2 年 9 月 30 日

ii

JaCVAM 運営委員会
平 林 容 子 （国立医薬品食品衛生研究所

安全性生物試験研究センター）：委員長

石 井 孝 司 （国立感染症研究所）
小川久美子 （国立医薬品食品衛生研究所

安全性生物試験研究センター病理部）

諫 田 泰 成 （国立医薬品食品衛生研究所

安全性生物試験研究センター

薬理部）

北嶋

安全性生物試験研究センター

毒性部）

聡 （国立医薬品食品衛生研究所

合 田 幸 広 （国立医薬品食品衛生研究所）
佐々木正広 （厚生労働省

医薬・生活衛生局

医薬品審査管理課

化学物質安全対策室）

杉 山 圭 一 （国立医薬品食品衛生研究所

安全性生物試験研究センター

変異遺伝部）

髙 橋 祐 次 （国立医薬品食品衛生研究所

安全性生物試験研究センター

毒性部

高 畑 正 浩 （厚生労働省

医薬・生活衛生局

医薬品審査管理課）

束 野 正 明 （厚生労働省

医薬・生活衛生局

医薬品審査管理課

広 瀬 明 彦 （国立医薬品食品衛生研究所
笛 木

医薬品医療機器総合機構）

横 田 雅 彦 （独立行政法人

医薬品医療機器総合機構）

足利太可雄 （国立医薬品食品衛生研究所

化学物質安全対策室）

安全性生物試験研究センター

修 （独立行政法人

動物管理室）

安全性予測評価部）

安全性生物試験研究センター

安全性予測評価部

安全性生物試験研究センター

安全性予測評価部第二室）：事務局
小島肇 （国立医薬品食品衛生研究所第二室）：事務局

iii

JaCVAM statement on
the Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)
Skin Sensitization Test Method

At a meeting held on 25 June, 2020, at the National Institute of Health Sciences (NIHS) in
Kanagawa, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: Although it is possible to classify chemicals that yield positive results using thisIL-8 Luc assay test method as skin sensitizers, it is difficult to classify accurately
their skin sensitization strength. If a negative result is obtained, it is not possible
to judge as a non-skin sensitizer. Therefore, the IL-8 Luc assay test method is notsuitable for predicting skin sensitization potential on its own; in order to make asuitable assessment, the results of IL-8 Luc assay testing must be used with athorough understanding of the properties of each test chemical in combination
with other information as part of an integrated approach to testing and assessment(IATA). Furthermore, when using this test, thorough consideration must be
given to the applicability domain and to the judgement of inconclusive chemicals.
This statement was prepared, following the review prepared by the Skin Sensitization Test
JaCVAM Editorial Committee, to acknowledge that the results of a review and study by the
JaCVAM Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we propose the IL-8 Luc assay test method as a useful means for
estimating skin sensitization by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

December 2, 2020v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:

Mr. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement of Life
Sciences): Chairperson
Ms. Yoko Hirabayashi (Center for Biological Safety and Research: CBSR, National
Institute of Health Sciences: NIHS)
Mr. Morihiko Hirota (Japan Cosmetic Industry Association)
Mr. Yoshiaki Ikarashi (NIHS)
Mr. Takanori Ikeda (Japan Pharmaceutical Manufacturers Association)**Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Kunifumi Inawaka (Japan Chemical Industry Association)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology) *Mr. Yuji Ishii (CBSR, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)*
Mr. Fumihiro Kubo (Pharmaceuticals and Medical Devices Agency)*
Mr. Kenichi Masumura (Japanese Environmental Mutagen Society)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (CBSR, NIHS/ Saiseikai Utsunomiya Hospital)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Ms. Keiko Yamamoto (Pharmaceuticals and Medical Devices Agency)**
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2018 to 30th September 2020*: From 1st April 2018 to 31st March 2020**: From 1st April 2020 to 30th September 2020

vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Yukihiro Goda (NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, CBSR, NIHS)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, CBSR, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, CBSR, NIHS)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Masahiro Sasaki (Ministry of Health, Labour and Welfare)
Mr. Keiichi Sugiyama (Division of Genetics and Mutagenesis, CBSR, NIHS)
Mr. Masahiro Takahata (Ministry of Health, Labour and Welfare)
Mr. Yuhji Taquahashi (Animal Management Section of the Division of Toxicology,
CBSR, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Masahiko Yokota (Pharmaceuticals and Medical Devices Agency)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

皮膚感作性試験代替法
Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)

目次

評 価 会 議 報 告 書 -- ---------- ------- --------- ------- --------- ------- --------- ------- --------- ------- - 1
評 価 報 告 書 --- ------- --------- ------- --------- ------- --------- ------- --------- ------- --------- ------- 7

OECD KEY EVENT BASED TEST GUIDELINE 442E,
IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENTON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOMEPATHWAY FOR SKIN SENSITISATION------------------------------------------------------337

評価会議報告書
皮膚感作性試験代替法
Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)

JaCVAM 評価会議

令和 2 年（2020 年）6 月 25 日

11

JaCVAM 評価会議
大 野 泰 雄 （公益財団法人 木原記念横浜生命科学振興財団）：座長
五十嵐良明 （国立医薬品食品衛生研究所）
池 田 孝 則 （日本製薬工業協会）**
石 井 雄 二 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
稲 若 邦 文 （日本化学工業協会）
井 上 智 彰 （日本免疫毒性学会）*
今 井 教 安 （日本動物実験代替法学会）
岩瀬裕美子 （日本製薬工業協会）*
久 保 文 宏 （独立行政法人 医薬品医療機器総合機構）*
中村るりこ （独立行政法人 製品評価技術基盤機構）
西 川 秋 佳 （国立医薬品食品衛生研究所 病理部／済生会宇都宮病院）
西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
沼澤聡 （日本毒性学会）

平 林 容 子 （国立医薬品食品衛生研究所 安全性生物試験研究センター）
廣 田 衞 彦 （日本化粧品工業連合会）
増 村 健 一 （日本環境変異原学会）
山 本 恵 子 （独立行政法人 医薬品医療機器総合機構）**
横 関 博 雄 （日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 9 月 30 日*：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日**：令和 2 年 4 月 1 日～令和 2 年 9 月 30 日

22

Interleukin-8 Reporter Gene Assay（IL-8 Luc assay）は、化学物質の皮膚感作性を予測する試験法である。本試験法は感作性発現の Key event のひとつである、樹状細胞が活性化す
る際の IL-8 の発現亢進を指標として利用し、ヒト単球系培養細胞株に 2 種のレポーター
遺伝子を導入した THP-G8 細胞の IL-8 遺伝子の発現量の変化を測定することによって皮膚感作性の有無を判定する 1)。本試験法については、International Cooperation on Alternative
Test Methods（ICATM）等の支援のもと、Japanese Center for the Validation of Alternative
Methods（JaCVAM）の主導によってバリデーション研究が実施され 2)、第三者評価 3)が行われたのち、2018 年 6 月に経済協力開発機構 (Organization for Economic Co-operation andDevelopment: OECD) 試験法ガイドライン (Test Guideline: TG) 442E の ANNEX III として追記された 4)。JaCVAM 評価会議は、皮膚感作性試験資料編纂委員会により作成された皮膚感作性試験評価報告書 IL-8 Luc assay5）を基に本試験法の妥当性について検討した。
1. 試験法の定義
名称：Interleukin-8 Reporter Gene Assay（IL-8 Luc assay）
代替する対象毒性試験：モルモットを用いる皮膚感作性試験（OECD TG406）およびマウスを用いる局所リンパ節試験 [LLNA (OECD TG429)、LLNA:DA (OECD TG442A)、
LLNA:BrdU-ELISA or -FCM (OECD TG442B)]
試験法の概略：本試験法では、樹状細胞のモデルとしてヒト単球系培養細胞株である THP1 細胞にルシフェラーゼレポーター遺伝子として IL-8 プロモーターで制御される Stableluciferase orange（SLO）遺伝子と Glyceraldehyde 3-phosphate dehydrogenase（GAPDH）プロ
モーターで制御される Stable luciferase red（SLR）遺伝子を導入した THP-G8 細胞を用い、被験物質を添加して培養後、SLO（λmax = 580 nm）および SLR（λmax = 630 nm）の発光をそれぞれに対応したフィルターを装備したルミノメーターで分別測定し、増加した
IL-8 プロモーター活性の程度により感作性の有無を判定する 6)。なお、SLR 遺伝子の発現量は細胞生存率の評価に加え、SLO 発現量の補正に利用する。
2. 評価に用いた資料および評価内容の科学的妥当性
本試験法は、皮膚感作性発現機序における重要なステップである抗原提示細胞の活性化を、その際に認められる IL-8 の発現上昇をもって測定するという点で、科学的に妥当である。感作成立には局所における好中球の動員が必須であり、IL-8 は代表的な好中球走化性因子であることが報告されている 7) 。また、IL-8 遺伝子のプロモーター配列に結合する転写因子 Nuclear Factor kB は種々の免疫系細胞の分化・活性化や炎症応答を制御するメ
ディエーターとして知られ、IL-8 等のサイトカイン分泌制御の他、樹状細胞の活性化に関与することも知られている 8)。
本試験法は、ICATM 等の支援による JaCVAM によるバリデーション研究とそれに続く第三者評価により、皮膚感作性ハザードを予測する試験の実施と評価のための戦略的統合
方式（Integrated Approach to Testing and Assessment: IATA）の一部として受け入れられるべきと報告されており 2,3)、2018 年 6 月に OECD TG に追記されている 4)。JaCVAM 皮膚感

33

作性試験資料編纂委員会では、現在までに公開されている情報を基に本試験法の皮膚感作性試験代替法としての科学的妥当性について評価し、ICATM 等の支援による第三者評価
結果 3）と同様に、証拠の重み付けや他の試験法との組み合わせで用いることを推奨している 4）。
3. 本試験法の有用性と適用限界
本試験法は、細胞株を用いる in vitro 試験法であり、動物実験における 3Rs の精神に合致している。試験に用いる THP-G8 細胞は市販されているが、株式会社ジーピーシー研究所と譲渡契約を締結する必要がある。アッセイは簡便で、抗体染色の操作がなく 16 時間で終了する。
本試験は液相での反応を必要とするため、被験物質が試験溶液調製用の媒体 X-VIVOTM
15 に 20 mg/mL の濃度で溶解しないものは陰性反応を示したとしても、そのまま陰性として判定することはできない。よって、こうした物質は判定不可物質として、バリデーショ
ン研究では一致性の評価から外している。また試験法の原理上、ルシフェラーゼ活性を阻害する物質は測定を妨害する可能性がある。さらに本試験法では、酸無水物では偽陰性率が高く、界面活性剤は偽陽性となることが多い。以上のことより、こうした物質は本試験法の適用範囲外物質として、バリデーション研究の一致性評価から除外された。
これを踏まえた、OECD TG 442E ANNEX III に記載されているバリデーション研究の結果によると、3 施設における施設内再現性は平均すると 87.7%（14 物質）、施設間再現性は87.5%（32 物質）であった 2)。X-VIVOTM 15 を用い、OECD TG 442E ANNEX III 収載の予測モデルで評価した結果、LLNA のデータがある 143 物質について、判定不可の 25 物質
を除くと、感度 96% (92/96)、特異度 41% (9/22)、正確度 86% (101/118)であった。また別に、適用範囲外に該当する 7 物質および判定不可の 23 物質を除いた 113 物質についての
結果は、感度 96% (92/96)、特異度 53% (9/17)、正確度 89% (101/113)であった 2)。
また本試験法において、プロハプテンおよびプレハプテンは適用範囲外ではないが、感作性発現に酸化や細胞による代謝が必要なこうした物質は偽陰性を生じる可能性がある。
さらに陰性反応を示した物質の場合、判定不可物質に該当するかどうかは目視確認での溶媒への溶解性の判断に依存するため、注意が必要である。
4. 目的とする物質又は製品の毒性を評価する試験法としての、社会的受け入れ性および行政上の利用の可能性
社会的受け入れ性：
本試験法は、細胞培養の技術と本試験に対応したフィルターを装備したルミノメーターがあれば容易に実施可能である。
本試験法の実施に必要な THP-G8 細胞は市販されており、試験実施に必要な消耗品費は
h-CLAT や U-SENSTM とほぼ同程度である（ただし細胞購入の費用は別）。本試験法は、生きた動物を用いないという点で 3Rs の精神に合致しており、社会的受け入れ性は高い。

44

行政上の利用性：
本試験法において陽性の結果が得られた場合、皮膚感作性物質と判定することは可能であるが、感作性の強度を分類することは困難である。また、陰性の結果が得られた場
合、非皮膚感作性物質と判定できない。したがって本試験法単独での皮膚感作性の判定は不十分であり、被験物質の特性を十分に理解した上で、IATA を構成するその他の情報と組み合わせて適切に評価することが必要である。なお、本試験法の利用にあたっては、適用範囲および判定不可物質の判断に十分に配慮した上で使用されるべきである。

参考文献（最終アクセス日：2020 年 8 月 17 日）
1)Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, Aiba S. (2011) An in
vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THPG8. Toxicol Sci. Dec;124:359-69.

2)

OECD (2017). Validation report for the international validation study on the IL-8 Luc
assay as a test evaluating the skin sensitizing potential of chemicals conducted by the IL8 Luc Assay. Series on Testing and Assessment No. 267, ENV/JM/MONO (2017)19.
Organization for Economic Cooperation and Development, Paris. Available at:https://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm

3)

Report of the peer review panel for the IL-8 Luciferase (IL-8 Luc) assay for in vitro skinsensitization. Series on testing and assessment. No.268 ENV/JM/MONO(2017)20.
Organization for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm

4)OECD TG442E (2018) In vitro skin sensitization assays addressing the key event on activationof dendritic cells on the adverse outcome pathway for skin sentitisation. Accessible at:
http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicalssection-4-health-effects_20745788

5)

JaCVAM 皮膚感作性資料編纂委員会：皮膚感作性試験報告書 Interleukin-8 Reporter
Gene Assay（IL-8 Luc assay）（2019 年 11 月 1 日）

6)

Kimura Y, Watanabe M, Suzuki N, Iwaki T, Yamakage K, Saito K, Nakajima Y, Fujimura C,
Ohmiya Y, Omori T, Kojima H, Aiba S. (2018) The performance of an in vitro skin sensitisation
test, IL-8 Luc assay (OECD442E), and the integrated approach with direct peptide reactive
assay (DPRA), J. Toxicol. Sci., 43 (12), 741-749.

7)

Weber FC1, Németh T, Csepregi JZ, Dudeck A, Roers A, Ozsvári B, Oswald E, Puskás LG,
Jakob T, Mócsai A, Martin SF. (2015) Neutrophils are required for both the sensitization andelicitation phase of contact hypersensitivity, J. Exp. Med., 212 (1), 15-22.

8)

Liu T, Zhang L, Joo D, Sun SC. (2017) NF-κB signaling in inflammation, Signal Transduct
Target Ther. 2, 17023.

55評価報告書
皮膚感作性試験代替法
Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)

皮膚感作性試験資料編纂委員会

令和元年（2019 年）11 月 1 日

17

皮膚感作性試験資料編纂委員会
小 島 幸 一 （委員長：一般財団法人

食品薬品安全センター）

足利太可雄 （国立医薬品食品衛生研究所）
安 達 玲 子 （国立医薬品食品衛生研究所）
佐 藤 一 博 （国立大学法人

福井大学）

瀬 崎 拓 人 （三井化学株式会社）
武 吉 正 博 （一般財団法人

化学物質評価研究機構）

福 山 朋 季 （麻布大学､（前）一般財団法人

残留農薬研究所）

2
8

略語
AOP:

Adverse Outcome Pathway

AP-1:

Activator Protein 1

ARE:

Antioxidant/electrophile Response Element

ARE-Nrf2:

ARE-Nuclear factor-erythroid 2-related factor 2

BrdU:

5-Bromo-2-deoxyuridine

CRO:

Contract Research Organization

DC:

Dendritic Cell

DMSO:

Dimethyl sulfoxide

DPRA:

Direct Peptide Reactivity Assay

EU:

European Union

FBS:

Fetal Bovine SerumGAPDH:

GlycerAldehyde 3-Phosphate Dehydrogenase

IATA:

Integrated Approaches to Testing and Assessment

h-CLAT:

Human Cell Line Activation test

ICATM:

International Cooperation on Alternative Test Methods

IL-8:Interleukin-8

IL-8 Luc Assay:

Interleukin-8 Reporter Gene Assay

JaCVAM:

Japanese Centre for the Validation of Alternatives Methods

KE:

Key Event

KeratinoSens

TM:

The ARE-Nrf2 luciferase KeratinoSensTM test method

LA:

Lactic acid

LLNA:

Local Lymph Node Assay

LuSens:The ARE-Nrf2 luciferase LuSens test method

MTA:

Material Transfer Agreement

4-NBB:

4-NitroBenzyl Bromide

NFkB:

Nuclear Factor kB

OECD:

Organisation for Economic Co-operation and Development

QSAR:

Quantitative Structure-Activity Relationship

REACH:

Registration, Evaluation, Authorization and Restriction of Chemicals

SLO:

Stable Luciferase Orange

SLR:

Stable Luciferase Red

TG:
U-SENS

Test Guideline
TM:

U937 cell line activation Test

UN GHS:

United Nations Globally Harmonized System of Classification and Labellingof Chemicals

VMT:

Validation Management Team

3
9

要旨
皮膚感作性は化学物質の安全性評価において重要な評価項目であり、従来、モルモッ
トやマウスを用いた動物実験によって評価されてきた。近年 EU における欧州化学品規制（REACH）では、コンピューターを用いた定量的構造活性相関（QSAR）モデルや invitro 試験法による安全性評価が推奨されており、動物実験によって安全性が評価された成分を含む化粧品の販売が禁止されたことから、動物を用いない in vitro 試験法の開発が強く望まれている。
IL-8 Luc assay は、感作性発現機序における第三段階のイベントである樹状細胞が活性化する際の IL-8 の発現亢進を利用した in vitro 試験法であり、東北大学の相場らにより開発された。単球系培養細胞株である THP-1 細胞に 2 種のレポーター遺伝子を導入した THP-G8 細胞を用い、活性化に伴う細胞での IL-8 遺伝子の発現量の変化をルシフェ
ラーゼレポーターアッセイで測定することによって皮膚感作性の有無を判定する試験法である。IL-8 Luc assay は、経済協力開発機構（OECD）試験法ガイドライン（TG） 442E
ANNEX I の h-CLAT と類似の方法であり、同様に類似の方法である U-SENSTM（ANNEXII）と共に、TG 442E の ANNEX III として改定版にこれら 3 種の試験法が記載された。
本報告書は、JaCVAM の主導によって行われたバリデーション研究についてまとめられたバリデーション報告書、ICATM の支援等のもと行われた第三者評価報告書および試験開発者の投稿論文などをもとに試験手順をまとめ、有用性と限界を評価したものである。
本試験法は、バリデーション研究実施中に、使用する媒体や予測方法の改善が複数回行われた。これらをもとに作成された TG 442E ANNEX III によれば、14 物質を用いて3 施設で実施され、施設内再現性の平均は 87.7%であった。また、32 物質を用いて 3 施設で行われた施設間再現性は 87.5%であった。34 物質を用いた 3 施設によるバリデー
ション研究においては、感度は 75 から 87%、特異度は 90 から 100%、正確度は 79 から 91%であった。
本試験法では、液相での反応系を必要とし、媒体の X-VIVOTM 15 に 20 mg/mL の濃度で溶解せずに陰性と判定された物質では、判定不可となり、溶解しても偽陽性を起こす
可能性が高い。
さらに、IL-8 Luc assay は、無水物では偽陰性率が高く、界面活性剤は偽陽性となる可能性がある。細胞毒性を示す界面活性剤系の物質は適用範囲外である。ルシフェラー
ゼ活性を阻害する物質は測定を妨害する可能性がある。プレハプテンやプロハプテン
は、バリデーション研究では多くが正しく判定されたが、IL-8 Luc assay で用いる細胞の代謝能は限定的であるため偽陰性を生じる可能性がある。
以上の結果から、本試験法は、強度感作性物質（UN GHS 1A 分類）に比べて、軽度から中等度感作性物質（UN GHS 1B 分類）で偽陰性を生じやすい傾向にあるため、本法単独での感作性強度分類や UN GHS のサブカテゴリー分類への利用には適さない。なお、本法では 20mg/mL で X-VIVOTM 15 に溶解せず、陰性相当の結果となった場合は判定不可となるが、現行結果からは特に陰性物質で判定不可となる割合が高いことから、陰性
結果の判定に重点を置く場合には、注意が必要である。
410

本委員会は、上記の本試験法の様々な限界を勘案すると、本試験法単独では皮膚感作性の判定は不十分で有り、証拠の重み付けや他の試験法との組み合わせで用いることを推奨する。

511

1. 緒言皮膚感作性を評価することは化学物質の安全性評価において重要である。化学物質の皮膚での接触感作性のリスクを動物で予測する試験法として、モルモットを用いる
皮膚感作性試験（OECD TG 406）や、マウスを用いる局所リンパ節試験（LLNA, OECD TG
429）がある。LLNA には、[3H-Methyl]-thymidine 取込量を測定する方法以外に、ATP 量を測定する LLNA:DA*（OECD TG 442A）や BrdU 取込量を測定する LLNA: BrdU-ELISA†
および BrdU-FCM‡（OECD TG 442B）がある 1)。
EU において、REACH では、安全性評価においては QSAR モデルや in vitro 試験法の活用が推奨されており 2)、EU 化粧品規則（EC No 1223/2009）では、動物実験によって安全性が評価された成分を使用した化粧品の販売が禁止された 3)（2013 年 3 月全面施行）。
そのため、化学物質の皮膚感作性を判定する代替法の開発が強く求められている。
In chemico 試験法としてペプチド結合反応を利用した DPRA（OECD TG 442C）、in vitro
試験法として、ケラチノサイト細胞系の標的遺伝子を用いた KeratinoSensTM（OECD TG
442D Appendix IA）および LuSens（OECD TG 442D Appendix IB）、単球系培養細胞の活性化を利用した h-CLAT（OECD TG 442E ANNEX I）および U-SENS™（OECD TG 442E
ANNEX II）が OECD から TG として公表されている 1)。これら以外に、同じく単球系培
養細胞の活性化を利用した皮膚感作性試験の in vitro 法である IL-8 Luc assay が提案されて、JaCVAM 等によってバリデーション研究が行われた。
IL-8 Luc assay は、多くの皮膚感作性物質が樹状細胞を活性化することを利用し、ヒト単球系培養細胞である THP-1 細胞に 2 種のレポーター遺伝子を導入した THP-G8 細胞を用いて、活性化に伴う IL-8 の発現量の変化をルシフェラーゼレポーターアッセイにより測定する試験法で、東北大学の相場らにより開発された 4)。IL-8 Luc assay のバ
リデーション研究の結果 5) については、ICATM 等の支援による第三者評価 6) が完了し、2017 年 10 月に OECD の TG に追記された（OECD TG 442E ANNEX III）1)。
JaCVAM 皮膚感作性試験資料編纂委員会（以下、委員会）は、IL-8 Luc assay の皮膚感作性試験代替法としての科学的妥当性について、現在までに公開されている情報をもとに評価したので、その結果を報告する。
2. 試験法の原理
皮膚感作性は、ヒトでは接触皮膚炎、動物（齧歯類）では接触過敏症として知られる
化学物質の毒性の一つである。OECD がまとめた有害性発現経路（AOP）では、化学物
質による皮膚感作は次の 4 つの重要な事象（KE）から成るとされている 7)。
KE1:

化学物質とタンパク質のシステイン残基あるいはリジン残基との共有結合*LLNA:DA とは、リンパ球の増殖反応を adenosine triphosphate（ATP）を指標として定量する LLNA
†LLNA:BrdU-ELISAとは、リンパ球の増殖反応を非放射性標識の 5-bromo-2-deoxyuridine（BrdU）取り込み量を ELISA（enzyme-linked immunosorbent assay）で定量する LLNA
‡ LLNA:BrdU-FCM とは、リンパ球の増殖反応を非放射性標識の BrdU 取り込み量を FCM（flowcytometry method）で定量する LLNA

6
12

KE2:

ケラチノサイトにおける炎症性応答および ARE-dependent pathway による遺伝
子発現

KE3:

樹状細胞（DC）の活性化（特異的細胞表面マーカーの発現、ケモカインやサイト
カインの産生）KE4:リンパ節における T 細胞の増殖

IL-8 Luc assay は IL-8 遺伝子のプロモーターを用いたレポーター遺伝子アッセイであり、親細胞として DC 様細胞への分化能を有する THP-1 細胞を使用している。IL-8 遺伝子のプロモーター領域には NFkB、AP-1 等の応答配列があり、本試験系は DC 様細胞の IL-8 遺伝子プロモーターを介する転写活性化を指標とし、IL-8 の産生を間接的に捉える試験系と解釈できる。
本試験系で使用される THP-G8 細胞の親株である THP-1 細胞において、感作性物質の刺激により IL-8 産生が誘導されることが報告されている 8)。IL-8 は代表的な好中球
走化性因子であり、感作成立には局所における好中球の動員が必須であることが報告されていることから 9)、IL-8 は局所への好中球動員によって感作成立に関与しているものと考えられる。また、IL-8 遺伝子のプロモーター配列に存在する NFkB は種々の免疫系細胞の分化・活性化や炎症応答を制御するメディエーターとして知られ、IL-8 等のサイトカイン分泌制御の他、DC の活性化に関与することも知られている 10)。
以上のことから、IL-8 Luc assay は感作性 AOP における KE3:樹状細胞の活性化を検出する試験系と位置づけることができる。
本法で使用される THP-G8 細胞は、h-CLAT で使用される THP-1 細胞に IL-8 プロモー
ターで制御される SLO 遺伝子と GAPDH プロモーターで制御される SLR 遺伝子を安定的に導入した細胞であり、SLO（λmax = 580 nm）および SLR（λmax = 630 nm）の発光をそれぞれに対応したフィルターを装備したルミノメーターで分別測定して感作性の有無を判定する 11)。二種のルシフェラーゼレポーター遺伝子のうち、GAPDH プロモーター
で制御される SLR 遺伝子の発現量は細胞生存率の評価に加え、IL-8 プロモーター依存性 SLO 発現量の補正に利用される。
3. 試験手順／判定
IL-8 Luc assay の試験手順および判定は、被験物質を曝露した IL-8 プロモーターで制御される SLO 遺伝子と GAPDH プロモーターで制御される SLR 遺伝子を安定的に導入した THP-G8 細胞における SLO および SLR 遺伝子の発現量解析によって実施される。
3-1. THP-G8 細胞の準備、試薬および反応性確認試験
本法では、株式会社ジーピーシー研究所（鳥取）より供給されている THP-G8 細胞を使用する。細胞は、起眠後、継代数として 12 回もしくは 6 週間以内の使用が可能であり、細胞の増殖速度によるが、理論的にはかなりの被験物質の試験が可能と算出される。細胞培養培地としては、RPMI-1640 の基礎培地に牛胎仔血清（FBS：終濃度 10%）、Penicillin G（終濃度 100 units/mL）、Streptomycin（終濃度 100 μg/mL）、Amphotericin B（終713濃度 0.25 μg/mL）、Puromycin（終濃度 0.15 μg/mL）および G418（CAS:108321-42-2、終濃
度 300 μg/mL）を加えたものを使用する。試験開始前には本法における細胞の反応性確認を実施するべきである。反応性の確認は、細胞を起眠後 1 ～ 2 週間もしくは 2 ～ 4 回の継代後に陽性対照物質である 4-NBB（CAS:100-11-8、純度 99%以上）および陰性対照物質である LA（CAS:50-21-5、純度 85%以上）を用いて行う。4-NBB で陽性反応（IndIL8LA が 1.4 以上）、LA で陰性反応（Ind-IL8LA が 1.4 より低い）を示した細胞のみを本実験に使用する。
試験実施前に、THP-G8 を 2 ～ 5×105 cells/mL の細胞濃度で培養フラスコに播種し、48 時間もしくは 96 時間培養する。試験日に、1×106 cells/mL の濃度で 10%FBS 含有
RPMI-1640（抗生物質は入っていない）に再懸濁した THP-G8 細胞を 96 穴平底黒色プレートに 50 μL ずつ加える（各 well あたりの最終細胞数は 5×104 cells/well）。
3-2. 被験物質および対照物質の調製（図 1）
被験物質および対照物質は、試験当日に調製する。IL-8 Luc assay では、被験物質および陽性対照物質の調製媒体として X-VIVOTM 15（Lonza）の使用が推奨されている。ただし X-VIVOTM 15 を用いた場合の再現性を証明するデータが乏しい。また、DMSO と比較した場合、X-VIVOTM 15 の正確度は高いが感度が低い 10) ため、X-VIVOTM 15 の有用性判断には疑問の余地がある。ガイドラインでは、X-VIVOTM 15 以外の媒体（DMSO、水等）や培養液を使用する科学的に妥当な理由が存在する場合は、使用してもよいと記載されている。まず、20 mg の被験物質を 1 mL の X-VIVOTM 15 に溶解または懸濁し（20 mg/mL）、ボルテックスミキサーにて激しく撹拌した後、攪拌機を用いて最大 8 rpm、
室温（約 20℃）、30 分間撹拌する。上記工程でも溶解しない場合、被験物質が完全に溶解するか安定した分散状態が得られるまで超音波破砕を行う。被験物質が X-VIVOTM
15 に溶解する場合は、さらに X-VIVOTM 15 で 5 倍希釈したものを Stock solution（4mg/mL）として以降の試験に使用する。被験物質が X-VIVOTM 15 に溶解しない場合は、撹拌機でさらに 30 分以上攪拌した後、15,000 回転（約 20,000G）で 5 分間遠心分離し、得られた上清を Stock solution として以降の試験に使用する。
初回試験は、細胞毒性および感作性のある濃度を検出するために実施する。Stocksolution を X-VIVOTM 15 を用いて公比 2 で段階希釈して得られた 11 濃度を調製する。
得られた被験物質調製液を 50 μL/well ずつ、96 穴平底黒色プレートにまかれている細胞に添加する。被験物質が X-VIVOTM 15 に溶解する場合、被験物質の終濃度は 0.002 から 2 mg/mL となる。一方、被験物質が X-VIVOTM 15 に完全に溶解しない場合、被験物
質の終濃度は X-VIVOTM 15 に飽和した被験物質量に依存する。2 回目以降の試験では、1 回目の試験結果を踏まえて、Stock solution を CV05§ の 4 倍高い濃度とする。初回試験の最高濃度で Inh-GAPLA が 0.05 より低くならなかった場合は、2 回目以降の Stocksolution も初回試験と同じ濃度を使用する。
2 回目以降の試験では、Stock solution を X-VIVOTM 15 で 1.5 倍段階希釈する事で調§CV05 とは、Inh-GAPLA が 0.05 より低くなる最低濃度814製する。各試験、各濃度とも 4 well ずつ設定する。
4-NBB を推奨陽性対照物質とし、20 mg を 1.5 mL チューブ内で 1 mL の X-VIVOTM
15 と混合し、8 rpm 以下の撹拌機で 30 分以上攪拌した後、15,000 回転（約 20,000G）で
5 分間遠心分離する。得られた上清を以降の試験に使用する。X-VIVOTM 15 を用いて 8
倍希釈および 16 倍希釈し、50 μL の希釈液を同量の THP-G8 細胞とともに 96 穴平底黒色プレートに播種し（4 well/濃度）、攪拌した後 CO2 インキュベーター内で 16 時間反応
させ、ルシフェラーゼ活性を測定する。
LA を推奨陰性対照物質とし、20 mg を 1.5 mL チューブ内で 1 mL の X-VIVOTM 15 に溶解させる（20 mg/mL）。さらに X-VIVOTM 15 を用いて 10 倍希釈（2 mg/mL）および 20
倍希釈（1 mg/mL）し、50 μL の希釈液を同量の THP-G8 細胞とともに 96 穴平底黒色プレートに播種し（4 well/濃度）、攪拌した後 CO2 インキュベーター内で 16 時間反応させ、ルシフェラーゼ活性を測定する。
上記対照物質と同等の陽性・陰性反応を証明する背景データがある場合、それらの物
質を対照物質として使用しても構わない。ウェル間の被験物質交差汚染を防ぐため、プ
レートをシールするなどの処置を施す。

915

X-VIVO TM 15 に 20mg/mL で溶解する場合

1016

X-VIVO TM 15 に 20mg/mL で溶解しない場合

1117

図 1 被験物質および対照物質の調製
3-3. 被験物質および対照物質の曝露
3-2 で調製した被験物質および対照物質を細胞懸濁液と 96 穴平底黒色プレート内にて 1 : 1 で混合し、37±1°C、5% CO2 条件下にて 16±0.5 時間培養する。揮発性の高い被験物質は揮発により他の被験物質曝露区と交差汚染が生じる可能性があるので、その他の被験物質とは別の単独のプレートにて実験を行う。
被験物質ごとに感作性を予測するために 2 ～ 4 回の試験を行う。各試験は新たな被験物質調製液および細胞を用いて別日に行う。
3-4. ルシフェラーゼ活性測定
ルシフェラーゼ活性は光学フィルターを備えた 96 穴平底黒色プレート用ルミノメー
ターを用いて測定する。測定前にルミノメーターの校正および色の識別能を橙色および赤色発光ルシフェラーゼを用いて実施する必要がある。
事前に温められた 100 μL の Tripluc® ルシフェラーゼ測定溶液を被験物質、媒体ない
し対照物質を曝露した細胞に添加する。プレートは通常の実験室内環境下（約 20°C）で
10 分間振とうした後、ルミノメーターで測定する。生物発光は各 well 3 秒ずつフィル
ター存在下（F1）と、非存在下（F0）で測定する。測定結果をもとに下記の項目を算出する。
IL8LA：橙色ルシフェラーゼ活性値で、IL-8 プロモーター活性を示す。



GAPLA：赤色ルシフェラーゼ活性値で、GAPDH プロモーター活性（細胞生存率および細胞数）を示す。



nIL8LA：IL8LA を GAPLA で除した値で、細胞生存率もしくは細胞数をノーマライズした後の各曝露区における IL-8 プロモーター活性を示す。



Ind-IL8LA：被験物質を曝露した細胞における nIL8LA を無処理細胞の nIL8LA で1218

除した値で、被験物質曝露により増加した IL-8 プロモーター活性の程度を示す。


Inh-GAPLA：GAPLA の阻害値。被験物質を曝露した細胞における GAPLA を無処理細胞の GAPLA で除した値で、被験物質曝露による細胞毒性の程度を示す。

3-5. データの解析
測定毎に以下の基準で陽性ないし陰性を判断する。
Ind-IL8LA が 1.4 以上で 95%信頼限界の下限が 1.0 以上を示す被験物質を陽性と判断する。一方で、Ind-IL8LA が 1.4 より低い場合か、95%信頼限界の下限が 1.0 より低い場合、もしくはその両方の場合を陰性と判断する。
3-6. 予測方法
4 回の独立した試験において 2 度の陽性反応が認められた場合は感作性陽性と判断する。一方で、4 回の独立した試験において 3 度の陰性反応が認められた場合は感作性陰性と判断する。
表 1 陽性および陰性反応判断の基準

表 1 で陰性と判断された被験物質で、20 mg/mL 濃度にて X-VIVOTM 15 に溶解した被
験物質は図 2 のごとく陰性と判断するが、溶解しない被験物質については判定不可と
する。

13
19

図 2 感作性予測モデル
3-7. 試験成立の条件
IL-8 Luc assay においては、以下の条件が成立する必要がある。


試験毎に設定する陽性対照物質（4-NBB）の少なくとも 1 濃度で Ind-IL8LA が 5.0 より高い。



試験毎に設定する陰性対照物質（LA）の全ての濃度で Ind-IL8LA が 1.4 より低い。



対照区（細胞と Tripluc® はあり，被験物質は無処置）における GAPLA の値が媒体
対照区（50 μL/well の 10%FBS 含有 RPMI-1640 に 50 μL/well の X-VIVOTM 15）の 1/5より低いプレートは採用不可とする。



媒体対照区および被験物質曝露区の全ての濃度における Inh-GAPLA の値が 0.05 より低いプレートは採用不可とする。

3-8. 試験手順および判定方法に関する樹状細胞を指標とした他試験との比較
本試験法は樹状細胞の活性化を指標にした検査法であることから、同様に樹状細胞の活性化に着目した h-CLAT や U-SENS™ との比較が想定される。しかし、h-CLAT お
よび U-SENS™ が細胞表面抗原の発現に着目している一方、本法では IL-8 遺伝子の活性化を指標としているため、両者を単純に比較する事は困難である。
IL-8 Luc assay の最大の利点は、培養時間の短縮化と操作の簡便化にあると言える。
h-CLAT と U-SENS™ がそれぞれ 24 時間および 45 時間の培養を必要とするのに対し、
IL-8 Luc assay は 16 時間で終了し、操作も比較的単純である。細胞の維持にも一般的な培地を用いており、継代頻度や手間も h-CLAT および U-SENS™ と同等と考えられる。
一方、難点としては、本法の調製媒体として推奨されている X-VIVOTM 15 に溶解しない被験物質において、判定不可と判断される点が挙げられる。
4. 精度
Phase 0（技術移転性の評価）および Phase I、IIa、IIb、IIc、III、IV（6 つのバリデーション研究）が、JaCVAM 主導で行われ 5)、続いてバリデーション結果に関して ICATM 等の支援による第三者評価 6) を受けた。
4-1. 技術移転性
Phase 0 として、参加する A、B、C の 3 施設が主導施設から IL-8 Luc assay の説明を受け、感作性物質の 4-NBB、Tetramethylthiuram disulfide と非感作性物質の LA の 3 物
1420質をそれぞれ評価した（Lab A は 2 回、Lab B は 1 回、Lab C は 3 回）。合計 18 回の実験のうち、Lab A が UN GHS 1A である 4-NBB を非感作性物質と 1 回誤判定した以外は正
しく判定された。バリデーション実行委員会（VMT）は IL-8 Luc assay の技術移転性の結果を妥当なものとした 5)。
4-2. 再現性
施設間再現性を検討するために行われた Phase I において、感作性陰性の物質に関しては良好な結果であったが、陽性の物質に関しては偽陰性の結果が多く、陽性とする基準、濃度範囲、細胞と被験物質のインキュベーション時間、陽性対照物質の変更などを
VMT から求められた。Phase IIa から Phase III においてこれらに対する改善が図られた。
さらに、Phase IV において被験物質の培地中のタンパク質（FBS 等）への結合による被験物質ロスを減らし偽陰性を減らす目的で媒体を DMSO から X-VIVOTM 15 に変更した
結果、予測精度も向上することが明らかとなった。技術移転性の結果を除き、Appendix
1 の数値はこれらの改善後の結果である 5)。
次に、Phase 毎の改良のポイントと結果について述べる。Phase I は、原法の IL-8 Luc
assay プロトコルで行われた。3 施設の施設間再現性は 70%であった。Phase IIa では、希釈濃度・希釈率等が修正された。施設間再現性は 70％であった。Phase IIb は、培養時間等が修正された。施設間再現性は 86%、施設内再現性は 78%だった。Phase IIc は、
ルシフェラーゼ活性値のクライテリアが修正された。施設間再現性は 86%、施設内再現性は 71%だった。Phase III でもルシフェラーゼ活性値のクライテリアに修正が加えられ、施設間再現性は 95%だった。Phase IV では、予測性の向上を目的に溶媒が XVIVOTM-15 に変更された。施設間再現性は 91%、施設内再現性は 93%だった。ただしこれは 5 品のみの結果であり不十分と考えられたため、本評価書では、本試験法の再現性は DMSO と X-VIVOTM 15 の両方の結果より評価し、妥当と判断した。
バリデーション研究が多くの phase を経たことから、事前に先行バリデーション研究などでプロトコルの最適化を行っておくことの重要性が示唆された。
施設内と施設間の再現性はいずれも、適用 16 時間の Phase IIb、IIc、III（以上 DMSO
溶媒）および Phase IV（X-VIVOTM 15 溶媒）の結果をクライテリア 3 により評価して算出された。したがって、溶媒として異なる 2 種類（DMSO と X-VIVOTM 15）の結果を統合して再現性が評価された。なお、Phase IIb の結果はバリデーション研究終了後に改めてクライテリア 3 に基づいて評価された。
4-2-1. 施設内再現性
施設内再現性は Phase IIb, IIc およびⅣの結果（Appendix 1 参照）から算出された。施設内再現性は Lab A が 85.7%（12/14）、Lab B が 91.7%（11/12）、Lab C が 85.7%（12/14）、
平均 87.7%であり 5)、 VMT の受け入れ基準（85%）を満たしており、本委員会は妥当と
判断した。

15
21

4-2-2. 施設間再現性
施設間再現性は Phase IIb, IIc、III およびⅣの結果（Appendix 1 参照）から算出された。
施設間再現性は、87.5%（28/32）であり 5) 、VMT の受け入れ基準（80%）を満たしており、本委員会は妥当と判断した。
5. 正確度（感度および特異度）
バリデーション報告書 5) によると、34 物質（Phase IIb から Phase IV、Appendix 1）を用いて 3 施設で行われた結果では、感度は 75 から 87%、特異度は 90 から 100%、正確度は 79 から 91%であった。
主導施設は、DPRA、KeratinoSensTM および h-CLAT で検討された物質を網羅する 143
物質について、最終化されたプロトコル（ガイドライン収載）（媒体は X-VIVOTM 15）に従い、IL-8 Luc assay を行った。その結果を LLNA およびヒトのデータと比較した 5)。
143 物質中判定不可の 25 物質を除く 118 物質を LLNA のデータ 11) と比較すると、感度96%（92/96）、特異度 41%（9/22）、正確度 86%（101/118）であった。143 物質から適用範囲外の 7 物質を除いた 136 物質から、さらに判定不可の 23 物質を除いた 113 物質を
LLNA と比較すると、感度 96%（92/96）、特異度 53%（9/17）、正確度 89%（101/113）であった。
ヒトのデータ 5) がある 90 物質のうち、判定不可の 14 物質を除く 76 物質では、感度93%（54/58）、特異度 39%（7/18）、正確度 80%（61/76）であった。適用範囲外の 6 物質と判定不可の 13 物質を除く 71 物質では、感度 93%（54/58）、特異度 54%（7/13）、正確度
86%（61/71）であった。
6. 評価可能な物質の範囲
IL-8 Luc assay のバリデーション報告書 5) には主導施設において検討された 143 物質を用いた評価結果が示されている。これは、本法開発当初の媒体である DMSO と、評
価物質の溶解性の改善のために用いることとした媒体 X-VIVOTM 15 を用いた結果である。これらに加えて、VMT との議論の中で予測方法の改善（Option 1 から Option 2）などが行われ、バリデーション報告書中には X-VIVOTM 15 を用いた Option 2 の結果が合わせて表に載せられている。
予測モデルは以下のように定義された。Option 1 では、第 1 回から第 4 回までの実験で、2 回の陽性結果を示した場合は陽性と判定する。3 回の陰性結果を示した場合「Supposed negative」とする。この中で、第 1 回の実験で Inh-GAPLA＜0.8 の化学物質と、Inh-GAPLA≧0.8 で X-VIVOTM 15 に 20 mg /mL 可溶の物質は陰性と評価する。InhGAPLA≧0.8 で X-VIVOTM 15 に不溶であれば判定不可と評価する。
Option 2 では、第 1 回から第 4 回までの実験で、2 回の陽性結果を示した場合は陽性と判定する。3 回の陰性結果を示した場合 「Supposed negative」 とする。この中で、XVIVOTM 15 に 20 mg/mL の濃度で可溶であれば陰性と評価し、不溶であれば判定不可と評価する。
最終的に Option 2 が採用されたが、この予測モデルでは、被験物質が 20 mg/mL で溶1622

解するか否かを判定基準とし、どの実験室でも明快で容易に判断できることを挙げている。しかも、この予測モデルの採用結果をみると、原法の施設内および施設間再現性の判定と大きく乖離していないし、むしろ正確度と感度は有意に向上し、評価の性能は原法と有意な差はないとバリデーション報告書には記載されている。
この表の一部に誤植（ガイドラインの記載内容には影響しない）が認められたため、開発者に確認して修正したものが Appendix 2 である。143 物質のうち 25 物質（LLNA で陽性が 11 物質、陰性が 14 物質）が判定不可である。さらに、5 物質（LLNA ですべて陰性）は、いずれも細胞毒性を示す界面活性剤系の物質で適用範囲外である。なお、判定不可の中にある 2 物質（LLNA ですべて陽性）は酸無水物で、適用範囲外にも含まれている。
IL-8 Luc assay で評価している 143 物質の判定方法および媒体の変更に伴って判定の結果がどのように変化したのかを、バリデーション報告書 217 頁（Appendix 16. Table S2
Option 1）および 269 頁（Appendix 16. Table S2 Option 2）のデータをもとにまとめたのが
表 2 である。さらに、本評価書 Appendix 2 に載せた表の 143 物質の判定の推移を表 2の物質から適用範囲外の物質を除いて同様にまとめたのが表 3 である。
いずれの表を見ても、媒体を DMSO から X-VIVOTM 15 に変更したことによって感度、正確度、および特異度は大きく変わらなかった。
判定方法を Option 1（Op-1）から Option 2（Op-2）へと変えたことによって、感度がさらに高くなり、特異度は低くなっている。これは判定不可（ガイドライン上では陰性でも陽性でもなく“Should not be considered”とされ、バリデーション報告書の表中では“inconclusive”とされている）とされた物質数が分母から除外（適用外も同様）されたためである。このことにより、特に LLNA やヒトのデータで非感作性と評価されている物質が除外候補となる確率が高く、結果的に陰性物質の評価数が減少してしまっている。したがって、被験物質が感作性陰性であることを示すための試験においては、XVIVOTM 15 の使用が適切であるとする十分なデータがないと考えられる。

17
23

表2

IL-8 Luc assay で評価している 143 物質の判定の推移*
右記条件
での判定

DMSO

X-VIVOTM 15

Op-1

Op-2

Sen

82

92

92

92

Non-sen

25

15

9

4

Inconclusive

---

---

6

11

Sen

11

13

13

13

Non-sen

25

23

16

9

Inconclusive

---

---

7

14

正確度
(%)

75
(107/143)80(115/143)83(108/130)86(101/118)

感度
(%)

77
(82/107)86(92/107)91(92/101)96(92/96)

特異度
(%)

69
(25/36)64(23/36)55(16/29)41(9/22)LLNA
Sen(107 物質)

Non-sen(36 物質)*バリデーション報告書 217 頁および 269 頁の表データを使用、Sen：陽性、Non-sen：
陰性、Inconclusive：判定不可、小数第 1 位四捨五入

表3

Appendix 2 に載せた 143 物質の判定の推移*
右記条件
での判定

DMSO

X-VIVOTM 15(Op-1)**

Op-2

Sen

82

92

(92)

92

Non-sen

23

13

(6)

4

Out of AD

2

2

(2)

2

Inconclusive

---

---

(7)

9

Sen

7

8

(8)

8

Non-sen

24

23

(17)

9

Out of AD

5

5

(5)

5

Inconclusive

---

---

(6)

14

正確度
(%)

78
(106/136)85(115/136)(89)(109/123)

89(101/113)

感度
(%)

78
(82/105)87(92/105)(94)(92/98)96(92/96)

特異度
(%)

77
(24/31)74(23/31)(68)(17/25)53(9/17)LLNA

Sen(107 物質)

Non-sen(36 物質)*Appendix 2 のデータを使用、表 2 の物質から適用範囲外の物質数を除いて正確度等が求められている。Sen：陽性、Non-sen：陰性、Out of AD：適用範囲外、Inconclusive：判
定不可、小数第 1 位四捨五入、**開発者確認データ
1824

技術的限界として、液相での反応を必要とする試験系であるため、X-VIVOTM 15 に20 mg/mL の濃度で溶解しない陰性の物質は判定不能となる。現試験法では、完全に溶解しない場合、遠心で沈殿物を除き上清を適用するため、適用濃度が不明である。XVIVOTM 15 に 20 mg/mL の濃度で溶解しない物質を感作性陰性と判断するためには、完
全に溶解した溶液で評価をするか、上清中の被験物質濃度の測定をするなどの改良が望まれる。ただし、科学的根拠があれば他の溶媒も使用可能とされている。
バリデーション研究においては、酸無水物の偽陰性率が高かった。界面活性剤は、偽陽性となる可能性がある。さらに、ルシフェラーゼ活性に干渉する化合物は測定を妨害する可能性がある。以上より、酸無水物、界面活性剤およびルシフェラーゼ活性に干渉する化合物は適用範囲外である。プレハプテン（酸化により活性化される物質）やプロ
ハプテン（P450 等による代謝活性化を必要とする物質）に関しては、これまでの検討では多くが正しく判定された（プレハプテン 11/11、プロハプテン 6/6、およびプロ/プレハ
プテン 6/8）5)。しかし、本細胞の代謝能は限定的である 12) ため、未検討のプロハプテ
ンやプレハプテンについては偽陰性を生じる可能性がある。従って、プロハプテンやプレハプテンの可能性がある化合物の陰性結果の解釈は注意が必要である。
7. 有用性と限界
本法は細胞実験とルミノメーターを組み合わせた皮膚感作性試験法であり、ルミノ
メーターを保有し、その技術に習熟した施設であれば実施可能と思われる。ただし本試
験法は 2 色同時発光のため、分離するフィルターを装着することができる特殊なルミ
ノメーターが必要である。
本試験法は動物を用いない in vitro の手法であり、同じ目的のために実施される動物実験と比べ、より「動物の愛護及び管理に関する法律」および 3Rs の精神と合致している。
IL-8 Luc assay 実施の際に必要となる消耗品費については、1 アッセイ当たり約 2.7 万円と試算され、LLNA（同約 10 万円）より低額であり、h-CLAT（同約 2 万円）や U-SENSTM（同約 1.8 万円）とほぼ同程度であった。一方で、本試験法実施に必要な THP-G8 細胞は一般的な細胞バンクでは取り扱われておらず、現状では株式会社ジーピーシー研究所
のみから入手可能であることから、価格面で h-CLAT や U-SENSTM で使用される THP1 や U-937 と比較して割高になる可能性がある。さらに、入手には株式会社ジーピー
シー研究所と譲渡契約（MTA）を締結する必要があり、現行の MTA（2018/2/9 付）では本試験法を実施できるのは受託試験機関（CRO）以外で細胞培養機器を保有し、自ら本細胞を培養し試験用細胞ストックを作製可能な施設である。こうした点は試験実施の際に留意すべき点であると考えられる。試験期間については、IL-8 Luc assay は最短 4 日間で実施可能であり、LLNA（同 1 週間）、U-SENSTM（同 6 日間）や h-CLAT（同 6 日間）よ
り短期間であった。
本試験法は、試験の実施と評価のための戦略的統合方式（IATA）において他の試験法と組み合わせることにより、感作性物質と非感作性物質との区別に使用することが可能である 13)。しかし、強度感作性物質に比べて、軽度から中等度感作性物質で偽陰性の判
1925

定が生じやすい傾向にあるため、本法単独での感作性強度分類や UN GHS のサブカテゴ
リー分類への応用には適さない。
8. 結論
IL-8 Luc assay は、感作性発現機序における第三段階のイベントである樹状細胞が活性化する際の IL-8 の発現亢進を利用し、化学物質刺激に対する IL-8 誘導活性をレポー
ター遺伝子アッセイによって被験物質の皮膚感作性を評価する in vitro 試験法である。
本法で使用される THP-G8 細胞は、h-CLAT で使用される THP-1 細胞（単球系培養細胞）に IL-8 プロモーターで制御される SLO 遺伝子と GAPDH プロモーターで制御される SLR 遺伝子を安定的に導入した細胞であり、SLO（λmax = 580 nm）および SLR（λmax= 630 nm）の発光をそれぞれに対応したフィルターを装備したルミノメーターで分別測
定して感作性の有無を判定する。二種のルシフェラーゼレポーター遺伝子のうち、
GAPDH プロモーターで制御される SLR 遺伝子の発現量は、細胞生存率の評価に加え、IL-8 プロモーター依存性 SLO 発現量の補正に利用される。
本試験法は、バリデーション研究実施中に、使用する媒体や感作性予測モデルの改善などが頻繁に行われた。その結果をもとに作成された TG 442E ANNEX III によれば、
14 物質を用いて 3 施設で実施された施設内再現性の平均は 87.7%、32 物質を用いて 3
施設で実施された施設間再現性の平均は 87.5%であった。
TG 442E ANNEX III で示された媒体 X-VIVOTM 15 を用い、かつ TG 442E ANNEX III
収載の予測モデルによれば、LLNA のデータがある 143 物質について、判定不可の 25
物質を除いた 118 物質の評価では、感度 96%（92/96）、特異度 41%（9/22）、正確度 86％（101/118）であった。適用範囲外 7 物質および判定不可 23 物質を除いた 113 物質では、
感度 96%（92/96)、特異度 53%（9/17）、正確度 89%（101/113）であった。143 物質のうち、ヒトのデータがある 90 物質について、判定不可の 14 物質を除く 76 物質の評価では、
感度 93%（54/58）、特異度 39%（7/18）、正確度 80%（61/76）であった。適用範囲外 6 物質
および判定不可 13 物質を除いた 71 物質では、感度 93%（54/58）、特異度 54%（7/13）、
正確度 86%（61/71）であった。この 143 物質を見る限り、本法は様々な化学物質の皮膚
感作性の予測が可能であることが示されている。
IL-8 Luc assay の試験法は、液相での反応を必要とする試験系であるため、X-VIVOTM
15 に 20 mg/mL の濃度で溶解しない物質が陰性の場合は判定不可となる。X-VIVOTM 15を用いる場合、評価された陰性物質が少ないこと、DMSO と比較して感度、正確度および特異度に大きな違いは見られないことから、X-VIVOTM 15 は媒体として DMSO よ
りも常に優れているとは一概には言えないと本委員会は考えている。
界面活性剤等の細胞毒性を引き起こすような物質、酸無水物、ルシフェラーゼ活性に干渉する物質などは適用範囲外である。プレハプテンやプロハプテンに関しては、本法開発の段階では正しく判定されているが、本細胞の薬物代謝能は限定的であるため、活性化に代謝系を必要とする化学物質では、その感作性が正しく検出されない可能性があり、未検討のプロハプテンやプレハプテンについては偽陰性を生じる可能性もある。
本試験法は、強度感作性物質（UN GHS 1A 分類)に比べて、軽度から中等度感作性物
20
26

質（UN GHS 1B 分類)で偽陰性の判定が生じやすい傾向にあるため、本法単独での感作性強度分類や UN GHS のサブカテゴリー分類への利用には適さない。
なお、本法では 20mg/mL で X-VIVOTM 15 に溶解せず、陰性相当の結果となった場合
は、判定不可となるが、現行結果からは特に陰性物質で判定不可となる割合が高いことから、陰性結果の判定に重点を置く場合には本法の適用には注意が必要である。本委員会は、上記の本試験法の様々な限界を勘案すると、本試験法単独では皮膚感作性の判定は不十分であり、上記の点に留意して、証拠の重み付けや他の試験法との組み合わせで用いることを推奨する。
9. 利益相反(COI)について
特になし。

21
27

10. 引用文献（最終アクセス日：2019 年 11 月 1 日）
1) OECD Guidelines for the Testing of Chemicals, Section 4 , Available at: http://www.oecdilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-healtheffects_20745788
2) Corrigendum to Regulation (EC) No 1907/2006 of the European Parliament and of theCouncil of 18 December 2006 concerning the Registration, Evaluation, Authorisation andRestriction of Chemicals (REACH), establishing3) REGULATION (EC) No 1223/2009 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL of 30 November 2009 on cosmetic products (recast) (Text with EEA relevance) ,Availableat:https://ec.europa.eu/health/sites/health/files/endocrine_disruptors/docs/cosmetic_1223_200
9_regulation_en.pdf
4) Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, Aiba S. (2011) An in
vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THPG8. Toxicol Sci.; 124:359-69
5) OECD (2017). Validation report for the international validation study on the IL-8 Luc assay
as a test evaluating the skin sensitizing potential of chemicals conducted by the IL-8 Luc
Assay. Series on Testing and Assessment No. 267, ENV/JM/MONO (2017)19. Organizationfor
Economic
Cooperation
andDevelopment,
Paris.
Availableat:https://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm
6) Report of the peer review panel for the IL-8 Luciferase (IL-8 Luc) assay for in vitro skinsensitization. Series on testing and assessment. No.268 ENV/JM/MONO(2017)20.
Organization for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm
7) OECD (2014) The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent
Binding
toProteins,
OECD
Publishing,Paris,
Availableat:
http://dx.doi.org/10.1787/9789264221444-en
8) Nukada Y, Miyazawa M, Kosaka N, Ito Y, Sakaguchi H, Nishiyama N. (2008) Production of
IL-8 in THP-1 cells following contact allergen stimulation via mitogen-activated protein
kinase activation or tumor necrosis factor-alpha production. J Toxicol Sci., 33, 175–85.
9) Weber FC1, Németh T, Csepregi JZ, Dudeck A, Roers A, Ozsvári B, Oswald E, Puskás LG,
Jakob T, Mócsai A, Martin SF. (2015) Neutrophils are required for both the sensitization andelicitation phase of contact hypersensitivity, J. Exp. Med., 212 (1), 15-22.
10) Liu T, Zhang L, Joo D, Sun SC. (2017) NF-κB signaling in inflammation, Signal Transduct
Target Ther. 2, 17023.
11) Kimura Y, Watanabe M, Suzuki N, Iwaki T, Yamakage K, Saito K, Nakajima Y, Fujimura C,
Ohmiya Y, Omori T, Kojima H, Aiba S. (2018) The performance of an in vitro skinsensitisation test, IL-8 Luc assay (OECD442E), and the integrated approach with direct
peptide reactive assay (DPRA), J. Toxicol. Sci., 43 (12), 741-749.
12) Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y,
Nishiyama N, and Itagaki H. (2010) A comparative evaluation of in vitro skin sensitisation
tests: the human cell-line activation test （h-CLAT) versus the local lymph node assay（LLNA). Alternatives to laboratory animals : ATLA 38:275-84.
13) Guidance Document on the Reporting of Defined Approaches and Individual Information
Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin
Sensitisation, Annex 1 and Annex 2. Available at: http://www.oecd.org/chemicalsafety/riskassessment/iata-integrated-approaches-to-testing-and-assessment.htm
2228

Appendix 1

バリデーション研究 Phase IIb、IIc、III、IV の結果

Phase Chemicals RepeatⅡb 5 codedⅡc 5 coded

3 sets3 setsNo,
No. Chemical name

CAS No.

LLNA
EC3

GHS
Category

1

p-Benzoquinone

106-51-4

0.099

1A

2

1-Thioglycerol

96-27-5

3.6

1B

3

Benzyl cinnamate

103-41-3

18.4

1B

4

Diethyl maleate

141-05-9

5.8

1B

5

2,4-Dichloronitrobenzene

611-06-3

1

1,4-Phenylenediamine

106-50-3

0.00991A

2

2-Mercaptobenzothiazole

149-30-4

1.7

1A

3

Glycerol

56-81-5NC

4Isopropanol

67-63-0NC

5Methyl salicylate

119-36-8

NC

NC

Lab A
Judge

Lab B
Judge

Lab C
Judge

G(1/2)
P(2/2)
G(1/2)
P(4/4)
P(2/3)
P(3/3)
P(4/4)
P(3/3)
P(4/4)
N(0/3)
N(0/3)
N(0/3)
N(0/3)
N(0/3)
N(0/4)
P(2/2)
P(2/4)
P(3/4)
P(2/2)
P(2/2)
P(2/2)
N(0/3)
N(1/4)
N(1/4)
N(1/4)
N(0/3)
N(0/3)
N(0/3)
P(2/4)
N(0/3)

G(1/2)
P(2/2)
P(2/2)
P(4/4)
P(3/3)
P(2/2)
P(2/2)
P(2/2)
P(3/3)
N(0/4)
N(0/3)
N(0/3)
N(0/3)
G(0/2)
G(1/3)
P(2/4)
N(1/4)
P(2/2)
G(1/3)
P(2/2)
P(2/2)
N(0/4)
N(0/3)
N(1/4)
P(2/2)
P(2/2)
P(2/4)
N(0/3)
N(0/4)
N(1/4)

P(2/2)
P(2/2)
P(2/2)
P(4/4)
P(3/3)
P(3/3)
P(4/4)
P(3/3)
P(3/3)
N(0/3)
N(0/4)
N(1/4)
G(1/3)
N(0/4)
N(1/4)
N(1/4)
N(1/4)
P(2/3)
P(2/3)
P(2/2)
P(2/2)
N(0/4)
N(0/3)
N(0/4)
N(1/4)
P(2/4)
P(2/4)
N(0/3)
N(0/4)
N(0/3)

23
29

Ⅲ

Ⅳ

20 coded

5 coded

1 set

3 sets1

2,4-Dichloronitrobenzene

611-06-3

NC

N(1/4) N(0/3) N(0/3)

2

DNCB(1-Chloro-2,4dinitrobenzene)

97-00-7

0.06

1A

P(2/2)

P(2/2) P(2/2)

3

Glyoxal solution

107-22-2

0.8

1A

P(2/2)

G(1/3) P(2/2)

4

1,2-Benzisothiazol-3(2H)one

2634-33-5

8

1B

P(2/2)

P(2/2) P(2/2)

5

Citral5392-40-55

1B

P(2/2)

P(2/2) P(2/2)

6

4-Hydroxybenzoic acid

99-96-7NC

N(0/4) N(0/3) N(0/4)

7Beryllium sulphate7787-56-6

0.001

1A

N(1/4) N(0/3) N(0/3)

8Formaldehyde

50-00-00.61

1A

P(2/3)

P(2/2) P(2/3)

9Chlorpromazine HCL

69-09-0

0.14

1A

P(2/2)

P(2/2) P(2/2)

10Chloramine T

127-65-1

0.4

1A

P(2/3)

P(2/2) P(2/2)

11

Benzyl salicylate

118-58-1

2.9

1B

P(2/2)

P(2/2) P(2/3)

12Dihydroeugenol

2785-87-7

6.8

1B

P(2/3)

P(2/2) P(2/2)

13

Nickel chloride

7718-54-9

1B

P(2/2)

P(2/2) P(2/2)

14Imidazolidinyl urea

39236-46-9

24

1B

P(2/3)

P(2/3) P(2/2)

15

R(+)Limonene

5989-27-5

69

1B

P(2/2)

N(0/4) N(0/3)

16Methylmethacrylate

80-62-690

1B

N(0/3) N(0/4) N(0/4)

17

Benzyl alcohol

100-51-6NC

N(0/4) N(0/3) N(0/3)

18

Dimethyl isophthalate

1459-93-4

NC

N(0/3) N(0/4) N(1/4)

19

4-Aminobenzoic acid150-13-0NCN(0/3) N(0/3) N(0/4)

20

Xylene1330-20-7

95.8

NC

N(0/4) N(0/3) N(0/3)

1Ethyleneglycol
dimethacrylate

97-90-528

1B

2

4-Nitrobenzylbromide

100-11-8

0.05

1A

3

4-Allylanisole

140-67-0

18

1B

4

Ethylenediamine

107-15-3

2.2

1B

5

Salicylic acid

69-72-7

P: positive, N: negative, G: inconclusive

24
30

NC

P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/4)
P(2/3)
P(2/2)
N(0/3)
P(2/3)
N(1/4)

P(2/4)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
N(1/4)
N(1/4)
N(1/4)
N(1/4)
N(1/4)
N(0/3)

P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
P(2/2)
N(0/3)
N(1/4)
N(1/4)
N(1/4)
N(0/3)
N(0/3)

Appendix 2 ガイドラインに収載された IL-8 Luc assay により得られた 143 物質の評価
結果の比較
GHS

LLNA
Potency
category

IL-8 Luc
assay(DMSO)

IL-8 Luc
assay(X-VIVO)

IL-8 Luc assay(Test Guideline
protocol)(Op. 2)

1-Benzoylacetone (1-BA)

1A

Extremepositive

positive

positive

1-Chloromethylpyrene

1A

Extremepositive

positive

positive

1A

Extremepositive

positive

positive

1A

Extremepositive

positive

positive

Bandrowski's base

1A

Extremepositive

positive

positive

7787-56-6

Beryllium sulfate tetrahydrate

1A

Extremenegative

positive

positive

770-34-8

Dinitrofluorobenzene

1A

Extremepositive

positive

positive

8886-38-4

Diphenylcyclopropenone(DPCP)

1A

Extremepositive

positive

positive

915646-46-5

Oxazolone

1A

Extremenegative

positive

positive10

106-51-4

p-Benzoquinone

1A

Extremepositive

positive

positive11

7778-50-9

Potassium dichromate

1A

Extremepositive

positive

positive

121154-59-2

Tetrachlorosalicylanilide

1A

Extremepositive

positive

positive13

123-31-9

1,4-Dihydroquinone

1A

Strongpositive

positive

positive14

106-50-3

1,4-Phenylenediamine(PPD)

1A

Strongnegative

positive

positive15

95-55-6

2-Aminophenol

1A

Strongpositive

positive

positive16

5307-14-2

2-Nitro-1,4-phenylenediamine

1A

Strongpositive

negativeinconclusive

17

94-36-0

Benzoyl peroxide

1A

Strongpositive

negativeinconclusive

18

100-39-0

Benzyl bromide

1A

Strongpositive

positive

positive19

127-65-1

Chloramine-T

1A

Strongpositive

positive

positive20

69-09-0

Chlorpromazine hydrochloride

1A

Strongpositive

positive

positive

217646-79-9

Cobalt chloride hexahydrate

1A

Strongpositive

positive

positive22

50-00-0

Formaldehyde

1A

Strongpositive

positive

positive23

111-30-8

Glutaraldehyde

1A

Strongpositive

positive

positive

246259-76-3

Hexyl salicylate

1A

Strongpositive

negativeinconclusive

25

55406-53-6

Iodopropynyl butylcarbamate

1A

Strongpositive

positive

positive

26

1166-52-5

Lauryl gallate

1A

Strongpositive

positive

No.

CAS#

1

93-91-4

21086-00-6

3

97-00-7

4

100-11-8

520048-27-5

6

27
28

Chemical

2,4-Dinitrochlorobenzene(DNCB)
4-Nitrobenzyl bromide (4NBB)

108-31-6

Maleic anhydride

1A

Strong35691-65-7

Methyldibromoglutaronitrile(MDGN)

1A

Strongnegativeout of negativeout ofADAD

positiveinconclusive,
negativeout of AD

positive

positive

positive

negative

negativeinconclusive,
negativeout of AD

out

out

29

85-44-9Phthalic anhydride

1A

Strong30

121-79-9

Propyl gallate

1A

Strongpositive

positive

positive31

90-15-3

1-Naphthol

1A

Moderate

positive

positive

positive

32

579-07-7

1-Phenyl-1,2-propanedione

1A

Moderate

positive

positive

positive33

96-27-5

1-Thioglycerol(3-Mercapto1,2-propanediol)

1B

Moderate

positive

positive

positive

342634-33-5

1,2-Benzisothiazolin-3-one

1B

Moderate

positive

positive

positive

of AD

of AD

25
31

IL-8 Luc
assay
(X-VIVO)

IL-8 Luc assay(Test Guideline
protocol)(Op. 2)

ChemicalGHS

3344-77-2

12-Bromo-1-dodecanol

1B

Moderate

positive

positive

positive36

818-61-1

2-Hydroxyethyl acrylate

1A

Moderate

negative

negative

negative37

149-30-4

2-Mercaptobenzothiazole (2MBT)

1A

Moderate

positive

positive

positive38

93-51-6

2-Methoxy-4-methylphenol

1B

Moderate

positive

positive

positive39

93-53-8

2-Phenylpropionaldehyde

1B

Moderate

positive

positive

positive40

4313-03-5

2,4-Heptadienal

1B

Moderate

positive

positive

positive41591-27-5

3-Aminophenol

1B

Moderate

positive

positive

positive42

109-55-7

3-Dimethylaminopropylamine

1B

Moderate

positive

positive

positive

4317369-59-4

3-Propylidenephthalide

1B

Moderate

positive

positive

positive44

119-84-6

3,4-Dihydrocoumarin

1B

Moderate

negative

positive

positive45

106-47-8

4-Chloroaniline

1B

Moderate

negative

positive

positive

46

21834-92-4

5-Methyl-2-phenyl-2-hexenal

1B

Moderate

positive

positive

positive47

101-39-3

a-Methylcinnamic aldehyde

1B

Moderate

positive

positive

positive48

118-58-1

Benzyl salicylate

1B

Moderate

positive

positive

positive49

122-57-6

Benzylideneacetone

1B

Moderate

positive

positive

positive50

104-55-2

Cinnamic aldehyde(Cinnamal)

1B

Moderate

positive

positive

positive51

23593-75-1

Clotrimazole

1B

Moderate

positive

negativeinconclusive52

141-05-9

Diethyl maleate

1B

Moderate

negative

positive

positive53

64-67-5

Diethyl sulfate

1B

Moderate

negative

positive

positive54

111-40-0

Diethylenetriamine

1B

Moderate

positive

positive

positive55

2785-87-7

Dihydroeugenol (2-Methoxy4-propylphenol)

1B

Moderate

positive

positive

Positive56

107-15-3Ethylenediamine

1B

Moderate

negative

positive

Positive57

107-22-2

Glyoxal1A

Moderate

negative

positive

Positive58

97-54-1

Isoeugenol

1A

Moderate

positive

positive

Positive59

111-80-8

Methyl-2-nonynoate

1B

Moderate

negative

positive

Positive60

2682-20-4

Methylisothiazolinone, 2Methyl-2H-Isothiazol-3-one

1A

Moderate

positive

positive

Positive

61

10101-97-0

Nickel sulphate hexahydrate

1B

Moderate

positive

positive

Positive

627718-54-9

Nickel (II) chloride

Moderate

positive

positive

Positive

63

18031-40-8

Perillaldehyde

1B

Moderate

positive

positive

Positive64

122-78-1

Phenylacetaldehyde

1B

Moderate

positive

positive

Positive65

108-46-3

Resorcinol

1B

Moderate

positive

positive

Positive66

133-37-9

Tartaric acid

1B

Moderate

positive

positive

Positive67

137-26-8

Tetramethylthiuramdisulphide(TMTD)

1B

Moderate

positive

positive

Positive

686728-26-3trans-2-Hexenal

1B

Moderate

positive

positive

Positive69552-30-7

Trimellitic anhydride

1B

Moderate

negative

positive

Positive

No.

CAS#

35

26
32

IL-8 Luc
assay(DMSO)

LLNA
Potency
category

IL-8 Luc
assay
(DMSO)

IL-8 Luc
assay(X-VIVO)

IL-8 Luc assay(Test Guideline
protocol)(Op. 2)No.

CAS#

Chemical

GHS

LLNA
Potency
category

70

112-45-8

Undec-10-enal

1B

Moderate

positive

positive

Positive715989-27-5(R)-(+)-Limonene

1B

Weak

negative

positive

Positive72

111-25-1

1-Bromohexane

1B

Weak

negative

negativeinconclusive73

103-11-7

2-Ethylhexyl acrylate

1B

Weak

positive

positive

Positive

74

431-03-8

2,3-Butanedione

1B

Weak

positive

positive

Positive75

140-67-0

4-Allylanisole

1B

Weak

negative

positive

Positive

7613706-86-0

5-Methyl-2,3-hexanedione

1B

Weak

negative

positive

Positive77514-10-3

Abietic acid

1B

Weak

positive

positive

Positive78

122-40-7

Amyl cinnamic aldehyde

1B

Weak

positive

positive

Positive79

62-53-3

Aniline1B

Weak

negative

negative

Negative80

94-09-7

Benzocaine

1B

Weak

negative

negativeinconclusive

81

103-41-3

Benzyl cinnamate

1B

Weak

positive

positive

Positive82

1565-94-2

Bis-GMA

1B

Weak

positive

positive

Positive

832426-08-6

Butyl glycidyl ether

1B

Weak

positive

positive

Positive

84

104-54-1

Cinnamic alcohol

1B

Weak

positive

positive

Positive

855392-40-5

Citral

1B

Weak

positive

positive

Positive86

103-95-7

Cyclamen aldehyde

1B

Weak

positive

positive

Positive87

97-90-5Ethylene glycol dimethacrylate

1B

Weak

negative

positive

Positive88

97-53-0

Eugenol

1B

Weakpositive

positive

Positive89

19317-11-4

Farnesal

1B

Weakpositive

positive

Positive90

106-24-1

Geraniol

1B

Weakpositive

positive

Positive91

101-86-0

Hexyl cinnamic aldehyde

1B

Weak

positive

positive

Positive92

107-75-5

Hydroxycitronellal

1B

Weak

positive

positive

Positive

9339236-46-9

Imidazolidinyl urea

1B

Weak

positive

positive

Positive94

80-54-6

Lilial

1B

Weak

positive

positive

Positive95

78-70-6

Linalool

1B

Weakpositive

positive

Positive

96

31906-04-4

Lyral

1B

Weak

positive

positive

Positive

97

80-62-6

Methyl methacrylate

1B

Weak

negative

negativeinconclusive98613-29-6

N,N-Dibutylaniline

1B

Weak

positive

positive

Positive99

112-05-0

Nonanoic acid

1B

Weak

positive

positive

Positive100

144-62-7

Oxalic acid

1B

Weak

positive

positive

Positive101

61-33-6

Penicillin G

1B

Weak

negative

negative

Negative

102

87-86-5

Pentachlorophenol

1B

Weak

positive

positive

Positive

1032050-14-8

Phenol, 2,2-azobis-

1B

Weak

positive

positive

Positive104

93-99-2

Phenyl benzoate

1B

Weak

positive

negativeinconclusive105

110-86-1

Pyridine

1B

Weaknegative

negative

Negative27
33

IL-8 Luc
assay
(X-VIVO)

IL-8 Luc assay(Test Guideline
protocol)(Op. 2)No.

CAS#

ChemicalGHS

1061330-20-7

Xylene1B

Weaknegative

negativeinconclusive

107

127-51-5α-iso-Methylionone

1B

Weak

positive

positive

Positive

108

109-65-9

1-BromobutaneNonnegative

negative

negativeinconclusive

109

71-36-3

1-ButanolNonnegative

negative

negative

Negative110874-23-7

2-Acetylcyclohexanone

Nonnegative

positive

positive

Positive111923-26-2

2-Hydroxypropyl methacrylateNonnegative

negative

negative

Negative112

611-06-3

2,4-Dichloro-1-nitrobenzeneNonnegative

negative

negativeinconclusive

113

99-96-7

4-Hydroxybenzoic acidNonnegative

negative

negativeinconclusive

114

92-48-8

6-MethylcoumarinNonnegative

negative

negativeinconclusive

100-06-1

Acetoanisole (4Methoxyacetophenone)Nonnegativeinconclusive

115

negative

negative

positiveout

positiveoutof AD

positiveout of AD

116

8001-54-5

Benzalkonium chloride

Non

negative117

65-85-0

Benzoic acidNonnegative

negative

negativeinconclusive

118

100-51-6

Benzyl alcohol

Nonnegative

negative

positive

Positive119108-90-7

ChlorobenzeneNonnegative

negative

negativeinconclusive

120

9004-54-0

DextranNonnegative

negative

negative

Negative121

84-66-2

Diethyl phthalateNonnegative

positive

positive

Positive122

68-12-2

Dimethyl formamide

Nonnegative

negative

negative

Negative

1231459-93-4

Dimethyl isophthalateNonnegative

negative

negativeinconclusive

124

94-02-0Ethyl benzoylacetate

Nonnegative

positive

negativeinconclusive

125

121-32-4Ethyl vanilline

Nonnegative

positive

positive

positive

126492-94-4

Furil

Non

negative

positive

negativeinconclusive

127

56-81-5GlycerolNonnegative

negative

negative

negative

128

57-09-0

Hexadecyltrimethylammonium
bromideNonnegative

positive

positive

129

67-63-0IsopropanolNonnegative130

50-21-5Lactic acidNonnegative131

119-36-8

Methyl salicylateNonnegative

of AD

out

out

of AD

positiveout of AD

negative

negative

negative

negative

negative

negativeinconclusiveof AD

negative

negative

positiveout

positiveoutof AD

positiveout of AD

132

124-07-2

Octanoic acid (Caprylic acid)Nonnegative133

150-13-0

p-Aminobenzoic acidNonnegative

negative

negativeinconclusive134

94-13-3

Propyl parabenNon

negative

negative

positive

positive

135

57-55-6

Propylene glycol

Nonnegative

negative

negative

negative

136

81-07-2

Saccharin

Nonnegative

negative

positive

positive137

69-72-7

Salicylic acidNonnegativeinconclusiveof AD

negative

negative

positiveout

positiveoutof AD

positiveout of AD

138

151-21-3

Sodium lauryl sulfate

Nonnegative139

3810-74-0

Streptomycin sulfate

Nonnegative

negative

negative

negative

140

63-74-1

Sulfanilamide

Nonnegative

negative

negativeinconclusive28
34

IL-8 Luc
assay(DMSO)

LLNA
Potency
categoryof ADIL-8 Luc assay(Test Guideline
protocol)(Op. 2)

IL-8 Luc
assay(DMSO)

IL-8 Luc
assay(X-VIVO)

positiveout

positiveoutof AD

positiveout of AD

No.

CAS#

Chemical

GHS

LLNA
Potency
category141

9005-65-6

Tween-80

Nonnegative142

121-33-5

VanillinNonnegative

positive

positive

positive

1437733-02-0

Zinc sulfate

Nonnegative

positive

positive

positiveof AD

out of AD: out of applicability domain, inconclusive: Should not be considered

29
35

OECD/OCDE

442E

Adopted:25 June 2018

KEY EVENT-BASED TEST GUIDELINE
IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY
EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE
ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATIONGENERAL INTRODUCTION
Activation of dendritic cells Key Event based Test Guideline
1. A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as
defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals(UN GHS) (1). There is general agreement on the key biological events underlying skin sensitisation. The
current knowledge of the chemical and biological mechanisms associated with skin sensitisation has beensummarised as an Adverse Outcome Pathway (AOP) (2), starting with the molecular initiating event
through intermediate events to the adverse effect, namely allergic contact dermatitis. In this instance, themolecular initiating event (i.e. the first key event) is the covalent binding of electrophilic substances tonucleophilic centres in skin proteins. The second key event in this AOP takes place in the keratinocytes
and includes inflammatory responses as well as changes in gene expression associated with specific cellsignalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The
third key event is the activation of dendritic cells (DC), typically assessed by expression of specific cellsurface markers, chemokines and cytokines. The fourth key event is T-cell activation and proliferation,
which is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (3).
2. This Test Guideline (TG) describes in vitro assays that address mechanisms described under the Key
Event on activation of dendritic cells of the AOP for skin sensitisation (2). The TG comprises test methods
to be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordancewith the UN GHS (1).
The test methods described in this TG are:- Human Cell Line Activation test (h-CLAT)- U937 cell line activation Test (U-SENS™)- Interleukin-8 Reporter Gene Assay (IL-8 Luc assay)© OECD, (2018)
You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.
In accordance with the decision of the Council on a delegation of authority to amend Annex I of the decision of the council on the Mutual
Acceptance of Data in the assessment of chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.

37

2│

442E

OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used togenerate the data and the readouts measured but can be used indiscriminately to address countries’
requirements for test results on the Key Event on activation of dendritic cells of the AOP for skinsensitisation while benefiting from the Mutual Acceptance of Data.

Background and principles of the test methods included in the Key Event based
Test Guideline
4. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classicalmethods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The
murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction response exclusively, and have also
gained acceptance, since they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; ARENrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals.
6. Test methods described in this TG either quantify the change in the expression of cell surface marker(s)associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
sensitisers have been reported to induce the expression of cell membrane markers such as CD40, CD54,
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
activation (2).
7. However, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficient
to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore data
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as nontesting methods, including read-across from chemical analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

© OECD 201838

OECD/OCDE

442E │ 3o the procedure used to
The term "test chemical" is used in this Test Guideline to refer to what is being tested1 and is not related
y to address 9.countries’
to forthe skin
applicability of the test methods to the testing of mono-constituent substances, multi-constituent
ls of the AOP
substances and/or mixtures. Limited information is currently available on the applicability of the test
methods to multi-constituent substances/mixtures (14) (15). The test methods are nevertheless technicallyapplicable to the testing of multi-constituent substances and mixtures. When considering testing ofy Event basedmixtures, difficult-to-test chemicals (e.g. unstable), or test chemicals not clearly within theapplicability domain described in this Guideline, upfront consideration should be given to whether
the results of such testing will yield results that are meaningful scientifically. Moreover, whenory animals. The classicaltesting multi-constituent substances or mixtures, consideration should be given to possible interference ofnusson and Kligman, andcytotoxic constituents with the observed responses.
of skin sensitisation. The
LLNA: DA (TG 442 A)
xclusively, and have also
terms of animal welfaren.

LITERATURE

ssing the first key event(OECD TG 442D; AREed for contributing
to the Nations UN (2015), Globally Harmonized System of Classification and Labelling of
1. United
Chemicals (GHS). Sixth revised edition. New York & Geneva: United Nations Publications. ISBN:
978-92-1-117006-1.
n of cell surface marker(s)Available at: [https://www.unece.org/trans/danger/publi/ghs/ghs_rev06/06files_e.html].
posure to sensitisers (e.g.
e activation ofSkin (2012), The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to
2. DC.
OECD
ers such as CD40, Proteins.
CD54, Part 1: Scientific Evidence. Series on Testing and Assessment No. 168. Available at:such as IL-1β and TNF-α,
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/12), associated withDC
PART1&docLanguage=En
3.

OECD (2010), The Local Lymph Node Assay. OECD Guideline for the Testing of Chemicals No.
429, Organisation for Economic Cooperation and Development, Paris. Available at:nsitisation AOP (2) (13),http://www.oecd.org/env/testguidelines
one may not be sufficientemicals. Therefore
data (1992), Skin Sensitisation. OECD Guideline for the Testing of Chemicals No. 406,
4. OECD
support the discrimination
Organisation for Economic Cooperation and Development, Paris. Available at:n used within Integrated
http://www.oecd.org/env/testguidelines
mplementary information,
5. OECD (2010), Skin Sensitisation: Local Lymph Node Assay: DA. OECD Guideline for the Testing oftion AOP as well as nonChemicals No. 442A, Organisation for Economic Cooperation and Development, Paris. Available at:ples of the use of data
http://www.oecd.org/env/testguidelines
ardised both in relation to
6.haveOECD
ive predictions,
been (2010), Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA. OECD Guideline for the
Testing of Chemicals No. 442B, Organisation for Economic Cooperation and Development, Paris.
Available at: http://www.oecd.org/env/testguidelines, neither to sub-categorise
7. OECD (2015), OECD Guideline for the Testing of Chemicals No. 442C: In Chemico Skin
authorities implementing
Sensitisation: Direct Peptide Reactivity Assay (DPRA). Paris, France: Organisation for Economicent decisions. However,
Cooperation and Development. Available at: http://www.oecd.org/env/testguidelines
methods may be used on
1

In June 2013, the Joint Meeting agreed that where possible, a more consistent use of theterm "test chemical" describing what is being tested should be applied in new and updated Test
Guidelines.

OECD2018
2018©©OECD

39

42 │

442E

OECD/OCDE

3. Thetest (2015),
methodsOECD
includedin thisfor
Test
differ No.
in relation
the procedureused to8.
OECD
Guideline
theGuideline
Testing ofmay
Chemicals
442D: IntoVitro
Skin Sensitisation:
generatethe dataLuciferase
and the readouts
measuredbut France:
can be Organisation
used indiscriminately
to address
countries’
ARE-Nrf2
Test Method.
Paris,
for Economic
Cooperation
andrequirements
for test
results at:onhttp://www.oecd.org/env/testguidelines
the Key Event on activation of dendritic cells of the AOP for skin
Development.
Availablesensitisation while benefiting from the Mutual Acceptance of Data.
9. Steinman RM. 1991. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol
9:271-96.
10. CauxC, Vanbervliet
Massacrier
M, Okumura
K, LanierBanchereau
Backgroundand B,
principles
of C,
theAzumatest methods
includedin the LL,
Key andEventbased J. 1994.
B70/B7-2
isidentical
to
CD86
and
is
the
major
functional
ligandfor
CD28
expressedon human
Test Guideline
dendritic cells. J Exp Med 180:1841-7.
4. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classical11.
Aibathat
S, Terunuma
A, ManomeH, andH. 1997.Test
Dendritic
cells
differently respondto haptens
methods
use guinea-pigs,
the Guinea
PigTagami
Maximisation(GPMT)of Magnusson
and Kligman,
andandirritantsby
theirproductionofcytokines
andexpressionof
co-stimulatory
molecules.
the Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation.Eur
TheJ
Immunol
27:3031-8.
murinetests, the
LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)
(5)
and
LLNA:
BrdU-ELISA(TG 442S,B)(6), allZU,
assessthe induction
response
exclusively,
andhave also
12. Aiba S, Manome H, Nakagawa
MollahMizuashi
M, Ohtani
T, YoshinoY, andTagami.
H.
gainedacceptance,
since
they
provideanadvantageovertheguinea
pig
tests
in
terms
of
animal2003. p38 mitogen-activated protein kinase and extracellular signal-regulated kinases play welfare
distinct
togetheran objective
the byinductionphase of skinhaptens,
sensitisation.
roleswithin theactivationmeasurementof dendritic ofcells
two representative
NiCl2 and DNCB. J Invest
Dermatol 120:390-8.
5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event
13.
OECD
Series Peptide
on Testing& Assessment
No. and256:second
Guidance
On TheOf(OECD
TG (2016).
442C; Direct
Reactivity
Assay (7)),
keyDocument
event (OECDTG Reporting
442D; AREDefined
ApproachesAnd
Individual
Information
SourcesTo
Be
Used
WithinIntegrated
Approaches
Nrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to the
To TestingAnd
Assessment(IATA)
Skin Sensitisation, Annex 1 and Annex 2.
evaluationof the skinsensitisation
hazardpotentialFor
of chemicals.
ENV/JM/HA(2016)29. Organisation for Economic Cooperation and Development, Paris. Available at:[https://community.oecd.org/community/iatass].
6. Testmethods described in this TG either quantify the change in the expression of cell surface marker(s)associatedwithT,theSakaguchi
process ofH,activation
monocytes
and DC M,
following
to sensitisers
14. Ashikaga
Sono S, ofKosaka
N, Ishikawa
Nukadaexposure
Y, Miyazawa
M, Ito(e.g.
Y,
CD54,
CD86)
or
the
changesin
IL-8
expression,acytokine
associatedwiththeactivation
of
DC.
Skin
NishiyamaN, Itagaki H. (2010), A comparative evaluation of in vitro skin sensitisation tests: thesensitisers
been activation
reported totest
inducethe expressionof localcell membranemarkers
such as CD40,
humanhavecell-line(h-CLAT)versus thelymph node
assay (LLNA).
Altern.CD54,
Lab.
CD80,
CD83,
and
CD86
in
additiontoinductionofproinflammatory
cytokines,
such
as
IL-1β
and TNF-α,
Anim. 38, 275-284.
and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
15.
Piroird,activation(2).C., Ovigne, J.M., Rousset, F., Martinozzi-Teissier, S., Gomes, C., Cotovio, J., Alépée, N.
(2015). The Myeloid U937 Skin Sensitization Test (U-SENS) addresses the activation of dendritic cell
event in theoutcomerepresents
pathway forskinsensitization.
In Vitro
29, 901-916.
7. However,as adverse
DC activation
only
one
key event Toxicol.
of the skinsensitisation
AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficient
to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore data
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as nontesting methods, including read-across from chemical analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

© OECD 201840

OECD/OCDE

442E │ 41

o the procedure used to
y to address countries’
APPENDIXII
ls of the AOP
for skin
PROFICIENCY SUBSTANCESy Event basedPrior to routine use of the test method described in this Annex to Test Guideline 442E, laboratories should

demonstrate technical proficiency by correctly obtaining the expected U-SENS™ prediction for the 10
substances recommended in Table 1 and by obtaining CV70 and EC150 values that fall within theory animals. The classicalrespective reference range for at least 8 out of the 10 proficiency substances. Proficiency substances were
nusson and Kligman, andselected to represent the range of responses for skin sensitisation hazards. Other selection criteria were thatof skin sensitisation. The
the substances are commercially available, and that high-quality in vivo reference data as well as high
LLNA: DA (TG 442 A)
quality in vitro data generated with the U-SENS™ method are available. Also, published reference data arexclusively, and have alsoavailable for the U-SENS™ method (1) (8).
terms of animal welfaren.
Table 1: Recommended substances for demonstrating technical proficiency with the U-SENS™ method

ssing the first key event(OECD TG 442D; AREed for contributing to theProficiency
substances

n of cell surface marker(s)posure to sensitisers (e.g.
e activation of DC.
Skin
4-Phenylenediamine
ers such as CD40, CD54,
such as IL-1β and TNF-α,
PicrylDC
sulfonic acid12), associated with
Diethyl maleate

nsitisation AOP (2) (13),one may not be sufficient
Resorcinol
emicals. Therefore data
support the discrimination
Cinnamic alcohol
n used within Integrated
mplementary information,
tion AOP as well4-Allylanisole
as nonples of the use of data
Saccharin
ardised both in relation
toive predictions, have been
Glycerol

CASRN

U-SENS™ U-SENS™

U-SENS™

PhysicalIn vivo
state prediction1

Solvent/
VehicleCV70
Reference
Range inµg/mL2

EC150
Reference
Range inμg/mL2106-50-3

Solid

Sensitiser(strong)

Completemedium3<30

Positive(≤10)

2508-19-2

LiquidSensitizer(strong)

Completemedium>50

Positive(≤50)

141-05-9

LiquidSensitiser(moderate)

DMSO

10-100

Positive(≤20)

108-46-3

Solid

Sensitiser(moderate)

Completemedium>100

Positive(≤50)

104-54-1

Solid

DMSO

>100

140-67-0

LiquidDMSO>10081-07-2

Solid

Non-sensitiser

DMSO

>200

56-81-5

LiquidNon-sensitiser

Sensitiser(weak)
Sensitiser(weak)

CompletemediumCompletemediumLactic acid50-21-5
Liquid Non-sensitiser, neither to sub-categorise
authorities implementing
Salicylic acid
69-72-7
Solid Non-sensitiser
DMSO
ent decisions. However,methods may Abbreviations:be used on CAS RN = Chemical Abstracts Service Registry Number
1

>200
>200
>200

Positive(10-100)
Positive(<200)
Negative(>200)
Negative(>200)
Negative(>200)
Negative(>200)The in vivo hazard and (potency) prediction is based on LLNA data (1) (8). The in vivo potency isderivedusing the criteria proposed by ECETOC (17).
2Based on historical observed values (1) (8).
3Complete medium: RPMI-1640 medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100
units/mL penicillin and 100 µg/mL streptomycin (8).
© OECD 2018
© OECD 2018

41

2 ││
42

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used togenerate the data and the readouts measured but can be used indiscriminately to address countries’
requirements for test results on the Key Event on activation of dendritic cells of the AOP for skin
ANNEX
III: IN VITRO
sensitisation
while benefitingfrom theSKIN
MutualSENSITISATION:
Acceptance of Data. IL-8 LUC ASSAY

Background and principles of the test methods included in the Key Event based
INITIAL
AND LIMITATIONSTestCONSIDERATIONSGuideline
4. The assessmentof skinsensitisation
has typicallyinvolvedof laboratory
animals.
The classical1.
In contrast
to assays
analysing
the expressionof thecellusesurface
markers,
the IL8-Luc
assay
methods thatuse guinea-pigs,
the Guinea
Pig Maximisationof Magnusson
and cells
Kligman,quantifies
changesin IL-8 expression,a cytokine
associatedTest
with(GPMT)
the activationof dendritic(DC).andIn
Buehler Test (TG(4), assessboth
the inductionand elicitation
phases acuteof skinsensitisation.
The
the THP-1-derived
IL-8406)
reportercell line(THP-G8,
establishedfrom the humanmonocytic
leukemiamurinethe LLNA(TG 429) (3)and its twofollowing
non-radioactive
modifications,
DAexpression(TG 442 A)
celllinetests,
THP-1),
IL-8 expression
is measured
exposure
to sensitisersLLNA:(1). Theof(5) and LLNA:(TG 442 B) (6),all assess
induction
exclusively, and have also
luciferaseis thenBrdU-ELISA
used to aid discrimination
betweenskin thesensitisers
andresponse
non-sensitisers.
gained acceptance, since they provide an advantage over the guinea pig tests in terms of animal welfare
together with
objective
measurementof evaluatedthe inductionof skinstudy
sensitisation.
2.
The anIL-8
Luc method
has been
in aphase
validation(2) conducted by the JapaneseCentre for the Validation of Alternatives Methods (JaCVAM), the Ministry of Economy, Trade and5. Recently
mechanistically-basedin Society
chemicofor
andAlternatives
in vitro test tomethods
the first
key eventIndustry(METI),and the JapaneseAnimaladdressing
Experiments(JSAAE)
and(OECD TG 442C;
DirectPeptide Reactivity
Assay(3)(7)),
andthesecondkey of
event
(OECD
TG
AREsubsequently
subjected
to independent
peer review
underauspices
JaCVAMandthe442D;
Ministryof
Nrf2 Luciferase
TestWelfare
Method(MHLW)(8)) of theskin
AOPhave been adopted
for contributing
to theHealth,Labour andwiththesensitisation
support of theInternational
Cooperation
on Alternativeevaluationof theskin sensitisation
hazardchemicals.
Test
Methods(ICATM).
Considering
all potential
availableofevidence
and input from regulators and stakeholders,the IL-8 Luc assay is considered useful as part of IATA to discriminate sensitisers from non-sensitisers for6. Testmethods
in this TG either
change inofthethe
expressionof cellLucsurfacethepurposeof describedhazard classificationand quantify
labelling.theExamplesuse of IL-8
assaymarker(s)data inassociatedwiththeprocessofactivationofmonocytes
andDC
following
exposuretosensitisers(e.g.
combination with other information are reported in the literature (4) (5) (6).
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
sensitisersThe
haveIL-8
beenreported
inducemembraneexperienced
markers such
CD40,
CD54,
3.
Luc
assay toprovedto thebe expression
transferableoftocell
laboratories
in as
cell
cultureandCD80, CD83,
and CD86 inWithin
additionandto induction
proinflammatory
cytokines, weresuch asIL-1β andand TNF-α,luciferase
measurement.
between oflaboratory
reproducibilities
87.7%
87.5%,and several (2).
chemokines
includingin IL-8
(CXCL8)study
and CCL3(10)published(11) (12),associatedwith thatDC
respectively
Data generated
the validation(2) and(9)
other
work
(1) (6) show
activation
versusthe (2).
LLNA, the IL-8 Luc assay judged 118 out of 143 chemicals as positive or negative and judged
25 chemicals as inconclusive and the accuracy of the IL-8 Luc assay in distinguishing skin sensitisers (UN
7. However,as DC
activation represents
one key
eventof the skin
AOP
(13),
GHS
Cat. 1) from
non-sensitisers(UN GHSonly
No Cat.)is 86%(101/118)with sensitisationa sensitivity of
96%(2)(92/96)
information
generatedwithtest
methods
measuringmarkersofDC
activation
alone
may
not
be
sufficientand specificity of 41% (9/22). Excluding substances outside the applicability domain described below
to conclude5),onthetheIL-8
presenceor absenceof skinpotentialof chemicals.
(paragraph
Luc assay
judged 113
out ofsensitisation
136 chemicals
as positiveor negative Therefore
and judgeddata
23
generated withthe test methods
described
proposed
to support
the discrimination
chemicals
as inconclusive
and theaccuracyinofthis
theTestIL-8Guideline
Luc assayare
is 89%(101/113)
with sensitivityof 96%
betweenandskinspecificity
sensitisersof (i.e.
GHSUsing
Category
andcited
non-sensitisers
usedthewithin
Integrated(92/96)53%UN(9/17).
human1)data
in Urbisch whenet al. (7),IL-8 Luc
assay
Approaches
toTesting
andAssessment(IATA),
togetherwithotherrelevant
complementary
information,
judged 76 out of 90 chemicals as positive or negative and judged 14 chemicals as inconclusive and thee.g. derived
from (61/76),
in vitro assays
addressing
keyandeventsof the skinsensitisation
AOP as well
as nonaccuracy
is 80%
sensitivity
is 93%other(54/58)specificity
is 39%(7/18). Excluding
substances
testing
methods,
includingread-acrossfromchemicalanalogues(13).
Examples
of
the
use
of
data
outside the applicability domain, the IL-8 Luc assay judged 71 out of 84 chemicals as positive or negative
generatedthese methods
within Defined
Approaches,
approaches
standardisedboth
relationtoandjudgedwith
13 chemicals
as inconclusive
and theaccuracy isi.e.
86%(61/71）withsensitivity
of in
93%
(54/58)thesetof
information
sources
used
and
in
the
procedureapplied
to
the
data
to
derivepredictions,
have
been
and specificity of 54% (7/13). False negative predictions with the IL-8 Luc assay are more likely to occur
published(13) andcan below/moderate
employed as usefulelements within
IATA.
with
chemicals
showingskin sensitisation
potency(UN GHS subcategory 1B) than thosewith high potency (UN GHS subcategory 1A) (6). Together, the information supports a role for the IL-8
8. Thetestinmethods
described in
Guidelinehazards.
cannot The
be usedon theirown,
to Lucsub-categorise
Luc
assay
the identificationof thisskinTest
sensitisation
accuracy
given
forneither
the IL-8
assay as askinsensitisers
into
subcategories
1A
and
1B
as
defined
by
UN
GHS
(1),
for
authorities
implementing
standalone test method is only for guidance, as the method should be considered in combination
with otherthese two
optional subcategories,
potency
for safetyassessment
decisions.
However,7
sourcesof informationin the contextnorof toan predict
IATA andin accordancewiththe provisionsof paragraphs
depending
the regulatory
framework,
positive
results
generatedwith thesemethods
maysensitisation,
be used on
and8 in theon
Generalintroduction.
Furthermore,whenevaluating
non-animal
methods
for skin
their
own
to
classifyachemical
into
UN
GHS
category1.
it should be remembered that the LLNA and other animal tests may not fully reflect the situation inhumans.

© OECD 2018
© OECD 201842

OECD/OCDE

442E │ 43

o the procedure used to
y to address 4.countries’On the basis of the data currently available, the IL-8 Luc assay was shown to be applicable to test
chemicalsls of the AOP
for skincovering a variety of organic functional groups, reaction mechanisms, skin sensitisation potency(as determined in in vivo studies) and physicochemical properties (2) (6).

5.
Although the IL-8 Luc assay uses X-VIVOTM 15 as a solvent, it correctly evaluated chemicals
TM
y Event basedwith a Log Ko/w >3.5 and those with a water solubility of around 100 µg/ mL as calculated by EPI Suiteand its performance to detect sensitisers with poor water solubility is better than that of the IL-8 Luc assay
using dimethyl sulfoxide (DMSO) as a solvent (2). However, negative results for test chemicals that are
ory animals. The
not classicaldissolved at 20 mg/ml may produce false negative results due to their inability to dissolve in Xnusson and Kligman,
VIVOTMand
15. Therefore, negative results for these chemicals should not be considered. A high falseof skin sensitisation.
negativeThe
rate for anhydrides was seen in the validation study. Furthermore, because of the limited
LLNA: DA (TG
442 A)
metaboliccapability of the cell line (8) and the experimental conditions, pro-haptens (substances requiring
xclusively, andhave also
metabolicactivation) and pre-haptens (substances activated by air oxidation) might give negative results interms of animal
welfareHowever, although negative results for suspected pre/prohaptens should be interpreted withthe assay.
n.
caution, the IL-8 Luc assay correctly judged 11 out of 11 pre-haptens, 6/6 pro-haptens, and 6/8 pre/prohaptens in the IL-8 Luc assay data set (2). Based on the recent comprehensive review on three non-animal
ssing the firstmethods
key event(the DPRA, the KeratinoSens™ and the h-CLAT) to detect pre and prohaptens (9), and based on(OECD TG 442D;
AREthe factthat THP-G8 cells used in the IL-8 Luc assay is a cell line derived from THP-1 that is used in theed for contributing
to theh-CLAT,
the IL-8 Luc assay may also contribute to increase the sensitivity of non-animal methods todetect pre and pro-haptens in the combination of other methods. Surfactants tested so far gave (false)
positive results irrespective of their type (e.g. cationic, anionic or on-ionic). Finally, chemicals thatn of cell surface
marker(s)interferewith luciferase can confound its activity/measurement, causing apparent inhibition or increased
posure to sensitisers(e.g. (10). For example, phytoestrogen concentrations higher than 1µM were reported to interfere
luminescencee activation of
DC.luminescence
Skin
with
signals in other luciferase-based reporter gene assays due to over-activation of theers such as CD40,
CD54,reporter gene. Consequently, luciferase expression obtained at high concentrations ofluciferase
such as IL-1β phytoestrogens
and TNF-α,or compounds suspected of producing phytoestrogen-like activation of the luciferase12), associatedwithDC
reporter gene needs to be examined carefully (11). Based on the above, surfactants, anhydrides andchemicals interfering with luciferase are outside the applicability domain of this assay. In cases where
there is evidence demonstrating the non-applicability of the IL-8 Luc assay to other specific categories of
nsitisation AOP(13), the method should not be used for those specific categories.
test(2)chemicals,
one may not be sufficientemicals. Thereforedata
6.
As described above, the IL-8 Luc assay supports discrimination of skin sensitisers from nonsupport the discrimination
sensitisers. Further work, preferably based on human data, is required to determine whether IL-8 Luc
n used withinresults
Integrated
can contribute to potency assessment when considered in combination with other information
mplementary information,
sources.
tion AOP as well as nonples of the use
7. of data
Definitions are provided in Appendix I.
ardised both in relation toive predictions, have been

PRINCIPLE OF THE TEST

, neither to sub-categorise
authorities implementing
8.
The IL-8 Luc assay makes use of a human monocytic leukemia cell line THP-1 that was obtainedent decisions.from
However,the American Type Culture Collection (Manassas, VA, USA). Using this cell line, the Dept. of
methods may Dermatology,
be used on Tohoku University School of Medicine, established a THP-1-derived IL-8 reporter cell line,
THP-G8, that harbours the Stable Luciferase Orange (SLO) and Stable Luciferase Red (SLR) luciferase
genes under the control of the IL-8 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
promoters, respectively (1). This allows quantitative measurement of luciferase gene induction by
detecting luminescence from well-established light producing luciferase substrates as an indicator of theactivity of the IL-8 and GAPDH in cells following exposure to sensitising chemicals.
© OECD 2018
© OECD 2018

43

2 ││
44

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used to
9.
The data
dual-colour
systemcomprises
luciferase (SLO;max countries’= 580 nm)
generate theand the assay
readouts
measuredbut ancanorange-emitting
be used indiscriminately
to address
(12)
for
the
gene
expression
of
the
IL-8
promoter
as
well
as
a
red-emitting
luciferase
(SLR;
max
630
requirements for test results on the Key Event on activation of dendritic cells of the AOP for =skin
nm)
(13)
for
the
gene
expressionoftheinternal
controlpromoter,GAPDH.
The
two
luciferases
emit
sensitisation while benefiting from the Mutual Acceptance of Data.
different colours upon reacting with firefly D-luciferin and their luminescence is measured simultaneously
in a one-step reaction by dividing the emission from the assay mixture using an optical filter (14)(Appendix
II).
Backgroundand principles of the test methods included in the Key Event based

Test Guideline

10.
THP-G8 cells are treated for 16 hours with the test chemical, after which SLO luciferase activity
4. The assessmentof skinhas typicallyinvolved
the useof laboratory
animals.
The GAPDHclassical(SLO-LA)
reflecting
IL-8 sensitisation
promoter activity
and SLR
luciferase
activity(SLR-LA)
reflecting
methods thatuse guinea-pigs,
theTo
Guineaof Magnusson
promoter
activity
are measured.
make Pig
the Maximisationabbreviations Testeasy (GPMT)
to understand,
SLO-LAandandKligman,
SLR-LA andare
the BuehlerasTest(TG and406)GAPLA,(4), assess
both the inductionand elicitation
phasesof skinsensitisation.
The
designatedIL8LA
respectively.
Table 1 givesa descriptionof theterms
associated withmurine tests,
the LLNA(3) andTheits measured
two non-radioactive
modifications,
LLNA:
DA (TG 442
A)luciferase
activityin the (TGIL-8 429)
Luc assay.
values are used
to calculate
the normalised
IL8LA
(5)
and
LLNA:
BrdU-ELISA
(TG
442
B)
(6),
all
assesstheinduction
response
exclusively,
and
have
also
(nIL8LA), which is the ratio of IL8LA to GAPLA; the induction of nIL8LA (Ind-IL8LA), which is thegainedsincetheyofprovide
an advantage values
over thepig tests
in termscellsof animal
ratio
of acceptance,
the arithmetic
means
quadruple-measuredof guinea
the nIL8LA
of THP-G8
treatedwelfare
with atogetherwithanobjective
measurementoftheinduction
phase
of
skin
sensitisation.
test chemical and the values of the nIL8LA of untreated THP-G8 cells; and the inhibition of GAPLA (InhGAPLA), which is the ratio of the arithmetic means of quadruple-measured values of the GAPLA of THP5. Recently
mechanistically-based
chemicoandofinthe
vitro
test methods
addressing
firstandkeyusedevent
G8cells treatedwith a test chemicalinandthe values
GAPLA
of untreated
THP-G8thecells,as(OECD
TG for442C;
Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; AREan
indicator
cytotoxicity.
Nrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals.
Table 1. Description of terms associated with the luciferase activity in the IL-8 Luc assay
6.Abbreviations
Test methods describedin this TG either quantify the change in the expression of cell surface marker(s)Definition
associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
GAPLA
SLR luciferase activity reflecting GAPDH promoter activity
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
IL8LA
SLOreported
luciferase
reflecting
IL-8 ofpromoter
activity markers such as CD40, CD54,
sensitisers have been
to activity
induce theexpression
cell membrane
nIL8LA/ GAPLA
CD80,
CD83, and IL8LA
CD86 inaddition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,
Ind-IL8LA
nIL8LAincludingof THP-G8
cells(CXCL8)
treated withof untreatedcells with DC
andseveral chemokines
IL-8
andchemicals
CCL3 (9)/ nIL8LA(10) (11)(12), associatedactivation (2).
Inh-GAPLAGAPLA of THP-G8 treated with chemicals / GAPLA of untreated cells
7. However, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information
generated
testconcentration
methods measuring
activation
alone may
not be <sufficientCV05
The with
lowestof the markers
chemicalofatDC
which
Inh-GAPLA
becomes
to conclude on thepresence or absence of skin sensitisation potential of chemicals. Therefore data
0.05.
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skinsensitisers(i.e. UN
1) andnon-sensitisers
whenofused
withinin Integrated
11.
Performancestandards(PS)GHS(15) Category
are available
to facilitate
the validation
modifiedvitro IL-8
Approaches
toTesting
andAssessment(IATA),
togetherwithotherrelevant
complementary
information,luciferase test methods similar to the IL-8 Luc assay and allow for timely amendment of
this Teste.g. derived
vitro assays
addressing
other of
keyDataeventsof thewill
skinsensitisation
AOPforas testwellmethods
as nonGuideline
forfromtheirininclusion.
Mutual
Acceptance
(MAD)
only
be guaranteed
testing
methods,
includingread-acrossfromchemicalanalogues(13).
Examples
of
the
use
of
data
validated according to the PS, if these test methods have been reviewed and included in this Test Guideline
generatedwith(16).
these methods within Defined Approaches, i.e. approaches standardised both in relation toby
the OECD
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods describedin this Test Guideline cannot be used on their own, neither to sub-categorise
DEMONSTRATIONOF PROFICIENCY

skin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these twoPrior
optional
subcategories,
nor test
to predict
forinsafetyassessment
decisions.
However,12.
to routine
use of themethod potency
described
this Annex
to Test
Guideline
442E,
depending onshould
the regulatory
framework,
positive
results using
generatedthese methods
may be listed
used onlaboratories
demonstratetechnical
proficiency,
the with
9 Proficiency
Substancesin
their own toa chemical
into
GHSincategory
1.
Appendix
IIIclassify
in compliance
with
theUN
Good
vitro Method
Practices (17). Moreover, test method users
should maintain a historical database of data generated with the reactivity checks (see paragraph 15) andwith the positive and solvent/vehicle controls (see paragraphs 21-24), and use these data to confirm thereproducibility of the test method in their laboratory is maintained over time.
© OECD 2018
© OECD 201844

OECD/OCDE

442E │ 45

o the procedure used to
y to address countries’
ls of the AOP for skin

PROCEDURE

13.
The Standard Operating Procedure (SOP) for the IL-8 Luc assay is available and should be
employed
when performing the test (18). Laboratories willing to perform the test can obtain they Event based
recombinant THP-G8 cell line from GPC Lab. Co. Ltd., Tottori, Japan, upon signing a Material Transfer
Agreement (MTA) in line with the conditions of the OECD template. The following paragraphs provide a
ory animals. The
classicalof the main components and procedures of the assay.
description
nusson and Kligman, andof skin sensitisation. Preparation
Theof cells
LLNA: DA (TG 442 A)
14.have also
The THP-G8 cell line from GPC Lab. Co. Ltd., Tottori, Japan, should be used for performing thexclusively, and
IL-8
Luc
assay
(see paragraphs 8 and 13). On receipt, cells are propagated (2-4 passages) and stored frozen
terms of animal welfareasahomogeneousstock. Cells from this stock can be propagated up to a maximum of 12 passages or an.
maximum of 6 weeks. The medium used for propagation is the RPMI-1640 culture medium containing
ssing the first10%
key foetal
event bovine serum (FBS), antibiotic/antimycotic solution (100U/mL of penicillin G, 100µg/mL of
streptomycin(OECD TG 442D;
ARE- and 0.25µg/mL of amphotericin B in 0.85% saline) (e.g. GIBCO Cat#15240-062),
0.15μg/mL
ed for contributing to thePuromycin (e.g. CAS:58-58-2) and 300μg/mL G418 (e.g. CAS:108321-42-2).

15.
Prior to use for testing, the cells should be qualified by conducting a reactivity check. This checkshouldben of cell surface marker(s)performed 1-2 weeks or 2-4 passages after thawing, using the positive control, 4-nitrobenzyl
bromide(e.g.
(4-NBB) (CAS:100-11-8, ≥ 99% purity) and the negative control, lactic acid (LA) (CAS:50-21-5,
posure to sensitisers≥85%
purity).
e activation of DC. Skin 4-NBB should produce a positive response to Ind-IL8LA (≥1.4), while LA should produce anegative
response to Ind-IL8LA (<1.4). Only cells that pass the reactivity check are used for the assay. The
ers such as CD40,
CD54,
check
shouldsuch as IL-1β and TNF-α, be performed according to the procedures described in paragraphs 22-24.

12), associated with DC
16.
For testing, THP-G8 cells are seeded at a density of 2 to 5 × 105 cells/mL, and pre-cultured inculture flasks for 48 to 96 hours. On the day of the test, cells harvested from the culture flask are washedwithcontaining 10% FBS without any antibiotics, and then, resuspended with RPMI-1640
nsitisation AOP(2)RPMI-1640(13),
containing
10%
FBS
without any antibiotics at 1 × 106 cells/mL. Then, cells are distributed into a 96-well
one may not be sufficient4flat-bottom
emicals. Therefore
datablack plate (e.g. Costar Cat#3603) with 50µL (5 × 10 cells/well).

support the discrimination
Preparation of the test chemical and control substances
n used within Integrated
mplementary information,
17.
The test chemical and control substances are prepared on the day of testing. For the IL-8
tion AOP as well
non-test chemicals are dissolved in X-VIVOTM 15, a commercially available serum-free medium
Luc asassay,
ples of the useof data(Lonza,04-418Q), to the final concentration of 20 mg/mL. X-VIVOTM 15 is added to 20 mg of test
ardised both inchemical
relation (regardlesstoof the chemical’s solubility) in a microcentrifuge tube and brought to a volume ofive predictions,haveandbeen
1mL
then vortexed vigorously and shaken on a rotor at a maximum speed of 8 rpm for 30 min at an
ambient temperature of about 20°C. Furthermore, if solid chemicals are still insoluble, the tube is sonicateduntil the chemical is dissolved completely or stably dispersed. For test chemicals soluble in X-VIVOTM, neither to sub-categorise
15, the solution is diluted by a factor of 5 with X-VIVOTM 15 and used as an X-VIVOTM 15 stock solution
authorities implementingof the test chemical (4 mg/mL). For test chemicals not soluble in X-VIVOTM 15, the mixture is rotatedent decisions.again
However,
for at least 30 min, then centrifuged at 15,000 rpm (≈20,000g) for 5 min; the resulting supernatant ismethods may used
be usedas anonX-VIVOTM 15 stock solution of the test chemical. A scientific rationale should be provided forthe use of other solvents, such as DMSO, water, or the culture medium. The detailed procedure fordissolving chemicals is shown in Appendix V. The X-VIVOTM 15 solutions described in paragraphs 18-23
are mixed 1:1 (v/v) with the cell suspensions prepared in a 96-well flat-bottom black plate (see paragraph16).
© OECD 2018
© OECD 2018

45

2 ││
46

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used to18.
The data
first andtest therun readouts
is aimedmeasured
to determine
the cytotoxic
concentration andto toaddress
examinethe skingenerate thebut canbe used indiscriminately
countries’TMTMsensitisingchemicals.
X-VIVO15, serialof dilutions
the ofX-VIVO
15 skinstock
requirementspotential
for testofresultson theUsing
Key Eventon activation
dendritic ofcells
the AOP for
solutions
of
the
test
chemicals
are
made
at
a
dilutionfactoroftwo(seeAppendixV)usinga
96-wellassay
sensitisation while benefiting from the Mutual Acceptance of Data.
block (e.g. Costar Cat#EW-01729-03). Next, 50 μl/well of diluted solution is added to 50 μl of the cellsuspension in a 96-well flat-bottom black plate. Thus for test chemicals that are soluble in X-VIVO TM 15,
the finalBackgroundconcentrations
the test chemicals
fromincluded
0.002 to in2 themg/mL(Appendix
andofprinciplesof thetestrange
methods
Key EventbasedV). For testTMchemicals
that
are
not
solublein
X-VIVO
15
at
20
mg/mL,
only
dilutionfactorsthat
rangefrom 2 to 210,
Test Guideline
are determined, although the actual final concentrations of the test chemicals remain uncertain and areTM
4. The assessmentof skin sensitisation
typically
involved inthetheuseof laboratory
animals.
The classicaldependent
on the saturated
concentrationhasof thetest chemicals
X-VIVO
15 stocksolution.
methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andTM
the Buehler(TG 406)
the induction
and fourth
elicitation
phases ofsensitisation.
The
19.
In Testsubsequenttest(4),runsassess(i.e. boththe second,
third, andreplicates),
theskin
X-VIVO
15 stockmurine tests,
the at
LLNA(TG 429) (3)4and
its higher
two non-radioactive
modifications,442 theA)
solution
is madethe concentration
times
than the concentrationof cellLLNA:
viabilityDA
05(TG(CV05;(5) andconcentration
LLNA: BrdU-ELISA
442 B) (6), allassess <0.05)
the induction
response
exclusively,
and have does
also
lowestat which (TGthe Inh-GAPLA
becomesin the firstexperiment.
If Inh-GAPLA
TM
gainedacceptance,they
advantage overthefirst
guineain terms
animalwelfarenotdecrease
below since
0.05 attheprovide
highest anconcentrationin therun, pig
the tests
X-VIVO
15ofstocksolutionistogetherwithanobjective
measurementoftheinduction
phase
of
skin
sensitisation.
made at the first run highest concentration. The concentration of CV05 is calculated by dividing theconcentration of the stock solution in the first run by dilution factor for CV05 (X) (dilution factor CV05
5. Recently
mechanistically-basedin chemicoand in tovitro
test(seemethods
addressing
key event(X);the dilution
factor required to dilute
stock solution
CV05)
Appendix
V). Forthe
testfirst
substancesnot(OECD inTG
442C; Direct
PeptideCV05
Reactivity
Assay (7)),
andconcentration
second key event
TG 442D;
AREsoluble
X-VIVO
at 20 mg/ml,
is determinedby theof the (OECDstock solution
x 1/X.
For
Nrf22 Luciferase
Teststock
Method(8)) isofprepared
the skin as
sensitisation
AOP have V).
been adopted for contributing to therun
to 4, a second
solution
4 x CV50 (Appendix
evaluation of the skin sensitisation hazard potential of chemicals.
20.
Serial dilutions of the X-VIVOTM 15 second stock solutions are made at a dilution factor of 1.5
6. Testa methods
described
in Next,
this TG
the solutionchange inexpression
surfacemarker(s)using
96-well assay
block.
50 either
μl/wellquantify
of diluted
is theadded
to 50 μl ofof cellthe cellsuspensioninassociatedwiththeprocessofactivationofmonocytes
andDC
following
exposuretosensitisers(e.g.
the wells of a 96-well flat-bottom black plate. Each concentration of each test chemical should be testedinor the changes
IL-8onexpression,a cytokine
associatedthe activationof DC.
Skin
4CD54,
wells.CD86)
The samples
are then in
mixed
a plate shaker
and incubatedforwith
16 hours
at 37°C and
5% CO
2,sensitisers
been reported
to isinducethe expressionof below.
cell membrane markers such as CD40, CD54,
afterwhichhavethe luciferase
activity
measuredas described
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,
TM
and several
including(CXCL8)andµL/well
CCL3 of(9)X-VIVO(10) (11)
(12),
with
DC
21.
Thechemokines
solvent control
is theIL-8
mixtureof 50
15andassociated50 µL/wellof cell
activation (2).
suspension
in RPMI-1640 containing 10% FBS.
7. However,DC activation
represents
onlyisone
key eventof ofthe4-NBB
skin sensitisation
(13),
22.
The asrecommended
positive
control
4-NBB.
20 mg
is preparedAOP
in a(2)
1.5-mL
TM
information
generatedwithtest
methods
measuringmarkersofDC
activation
alone
may
not
be
sufficient
microfuge tube, to which X-VIVO 15 is added up to 1 mL. The tube is vortexed vigorously and shaken
to conclude
the presenceof chemicals.
ona rotor at aonmaximum
speedorof absence
8 rpm forofatskinleast sensitisation
30 min. Afterpotential
centrifugation
at 20,000gTherefore
for 5 min,datatheTMgenerated withtest methods
described
in thisX-VIVO
Test Guideline
are proposed
supportthe discrimination
supernatantis thedilutedby a factorof 4 with
15, and500 μl ofto thedilutedsupernatant isTMbetween skin(i.e. UNGHS
Category
1) andnon-sensitisers
when
usedwithwithinIntegrated
transferred
to asensitisers
well in a 96-well
assay
block.
The diluted
supernatant
is further
dilutedX-VIVO
15
Approaches
Testing(IATA),
togetherwithotherrelevant
information,
atfactors of 2toand4, andand
50 Assessment
μl of the solution
is added
to 50
μl of
THP-G8cellcomplementarysuspension in thewells of ae.g. derived
from in black
vitro assays
addressingVI).
otherkeyconcentration
events of the ofskin
AOPshould
as wellbeastested
non96-wellflat-bottom
plate (Appendix
Eachthesensitisation
positive control
testing
methods,
includingread-acrossfromchemicalanalogues(13).
Examples
of
the
use
of
data
in 4 wells. The plate is agitated on a plateshaker, and incubated in a CO2 incubator for 16 hours
generatedthese
withinDefinedApproaches,
i.e. approaches(37°C,
5%withCO2),
aftermethods
which theluciferase
activity
is measured
as describedstandardisedin paragraphboth
29. in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
publishedThe(13)recommended
and can be employeduseful iselements
23.
negativeascontrol
LA. 20 within
mg of IATA.
LA prepared in a 1.5-mL microfuge tube, towhich X-VIVOTM 15 is added up to 1 mL (20 mg/ mL). Twenty mg/mL of LA solution is diluted by aTM
8. Theof
test
methods
described
Test Guideline
be usedLAon solution
their own,neither to sub-categorise
factor
5 withX-VIVO
15in(4thismg/mL);
500 μl of cannot
this 4 mg/mL
is transferred
to a well of aTMskin sensitisers
into subcategories
and 1BdefinedUNX-VIVO
GHS (1),forauthorities
implementing
96-wellassay block.
This solution is1Adilutedby asa factorof 2bywith
15 andthen diluted
again bythese
two
optional
subcategories,
nor
topredict
potencyforsafetyassessment
decisions.
However,a factor of 2 to produce 2 mg/mL and 1 mg/mL solutions. 50 μl of these 3 solutions and vehicle control
dependingTMon15)
theareregulatory
positivegenerated
may be usedon
(X-VIVO
added to framework,
50 µl of THP-G8cellresults
suspension
in thewith
wellstheseof a methods
96-well flat-bottom
black
their own
classify a chemical
UN GHS
category
1. in 4 wells. The plate is agitated on a plateshaker
plate.
Eachtoconcentrationof the into
negative
controlis tested
and incubated in a CO2 incubator for 16 hours (37°C, 5% CO2), after which the luciferase activity ismeasured as described in paragraph 29.

© OECD 2018
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OECD/OCDE

442E │ 47

o the procedure used to
y to address 24.
countries’Other suitable positive or negative controls may be used if historical data are available to derivecomparable
ls of the AOP
for skin run acceptance criteria.
25.
Care should be taken to avoid evaporation of volatile test chemicals and cross-contamination
between wells by test chemicals, e.g. by sealing the plate prior to the incubation with the test chemicals.

y Event based

26.
The test chemicals and solvent control require 2 to 4 runs to derive a positive or negative
prediction (see Table 2). Each run is performed on a different day with fresh X-VIVOTM 15 stock solution
ory animals. The
classicalof testchemicals and independently harvested cells. Cells may come from the same passage.
nusson and Kligman, andof skin sensitisation. The
LLNA: DA (TG 442 A)
activity measurements
xclusively, and have Luciferase
also
terms of animal welfare
27.
Luminescence is measured using a 96-well microplate luminometer equipped with optical filters,n.
e.g. Phelios (ATTO, Tokyo, Japan), Tristan 941 (Berthold, Bad Wildbad, Germany) and the ARVO series(PerkinElmer, Waltham, MA, USA). The luminometer must be calibrated for each test to ensure
ssing the first key event
reproducibility (19). Recombinant orange and red emitting luciferases are available for this calibration.
(OECD TG 442D; AREed for contributing to the28.
100µL of pre-warmed Tripluc® Luciferase assay reagent (Tripluc) is transferred to each well of
the plate containing the cell suspension treated with or without chemical. The plate is shaken for 10 min at
an ambient temperature of about 20°C. The plate is placed in the luminometer to measure the luciferase
n of cell surface marker(s)activity. Bioluminescence is measured for 3 sec each in the absence (F0) and presence (F1) of the opticalposure to sensitisers (e.g.
filter. Justification should be provided for the use of alternative settings, e.g. depending on the model ofe activation of DC. Skin
luminometer used.
ers such as CD40, CD54,
such as IL-1β and TNF-α,
29.
Parameters for each concentration are calculated from the measured values, e.g. IL8LA, GAPLA,12), associated with DC
nIL8LA, Ind-IL8LA, Inh-GAPLA, the mean ±SD of IL8LA, the mean ±SD of GAPLA, the mean ±SD of
nIL8LA, the mean ±SD of Ind-IL8LA, the mean ±SD of Inh-GAPLA, and the 95% confidence interval of
Ind-IL8LA. Definitions of the parameters used in this paragraph are provided in Appendices I and IV,
nsitisation AOP (2) (13),respectively.
one may not be sufficientemicals. Therefore data
30.
Prior to measurement, colour discrimination in multi-colour reporter assays is generally achieved
support the discrimination
using detectors (luminometer and plate reader) equipped with optical filters, such as sharp-cut (long-passn used within Integrated
or short-pass) filters or band-pass filters. The transmission coefficients of the filters for each
mplementary information,
bioluminescence signal colour should be calibrated prior to testing, per Appendix II.
tion AOP as well as nonples of the use of data
ardised both in relation toive predictions, have been

, neither to sub-categorise
authorities implementing
ent decisions. However,methods may be used on© OECD 2018
© OECD 2018

47

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48

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used to

DATA
REPORTING
generateAND
the data
and the readouts measured but can be used indiscriminately to address countries’
requirements for test results on the Key Event on activation of dendritic cells of the AOP for skin
Datawhileevaluation
sensitisation
benefiting from the Mutual Acceptance of Data.

31.
Criteria for a positive/negative decision require that in each run:- an IL-8
Luc assay prediction
is judged
positive
if a test chemical
Ind-IL8LA 1.4
and the lower
Backgroundand principlesof thetest methods
includedhas
in atheKey Event
basedlimit of the 95% confidence interval of Ind-IL8LA  1.0
Test Guideline- an IL-8 Luc assay prediction is judged negative if a test chemical has a Ind-IL8LA < 1.4 and/or the lower
4. Theofassessmentof skin sensitisation
has typicallyinvolved the use of laboratory animals. The classicallimit
the 95% confidence
interval of Ind-IL8LA< 1.0
methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The
murine Prediction
tests, the LLNA(TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)
model(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction response exclusively,
andth have alsord32.
Test chemicals
that provide
twoadvantage
positive results
among
the 1inst, terms
2nd, 3ofor
4 runsaregained acceptance,since they
provide anover thefrom
guineapig tests
animalwelfarestndrdth
identified
aspositives
whereas
those
that
give
three
negative
results
from
among
the
1
,
2
,
3
or
4
runs
together with an objective measurement of the induction phase of skin sensitisation.
are identified as supposed negative (Table 2). Among supposed negative chemicals, chemicals that are
dissolved
at 20
mg/ml of X-VOVOTM
are judged
as vitro
negative,chemicalsthat areat
5. Recently
mechanistically-basedin 15
chemicoand intest while
methods
addressing
the not
firstdissolved
key eventTM
20
mg/mlTGof 442C;X-VOVO
shouldReactivity
not be considered(Figure(OECD
Direct15
PeptideAssay (7)),
and 1).
second key event (OECD TG 442D; ARENrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals.
Table 2. Criteria for identifying positive and supposed negative

6. Testmethods describedin this TG 3rdeitherquantify the4th
changeexpressionof cell surface marker(s)1st run
2nd runrun
run in the Final
prediction
associatedwiththeprocessofactivationofmonocytes
andDC
following
exposure
Positive
Positive
Positive to sensitisers (e.g.
CD54, CD86) or the Negativechanges in IL-8 expression,
the activation of DC. Skin
Positive a cytokine- associated withPositive
sensitisers have been reported to induce the expression of cell membrane markers such as CD40, CD54,
Negative of proinflammatory
Positive
Positive
CD80, CD83, and CD86 in addition to induction
cytokines,
such as IL-1β and TNF-α,
Supposedand several chemokines including IL-8 (CXCL8) and Negative
CCL3 (9) (10) (11)(12), associated with DC
negative
activation (2).
Negative
Positive
Positive
Positive
7. However, as DC activation represents
only
one
key
event
of
the
skin
sensitisation AOP (2) (13),
Negative
Positive
Positive
information generated with test methods measuring markers
ofDC
activation
alone may not be sufficient
Negative
Supposed
to conclude on the presence or absence of skin sensitisation potential negativeof chemicals. Therefore data
generated with the testNegative
methods described
in
this
Test
Guideline
areproposed
to support the discrimination
Positive
Positive
Positive
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), togetherNegativewith other relevantSupposedcomplementary information,negative AOP as well as none.g. derived from in vitro assays addressing other key events of the skin sensitisation
Supposedof the use of data
testing methods, including read-acrossNegativefrom chemical analogues(13). Examples
generated with these methods within Defined Approaches, i.e. approaches negative
standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

© OECD 2018
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OECD/OCDE

442E │ 49

o the procedure used to
y to address countries’
Figure1. Prediction model for final judgment
ls of the AOP
for skiny Event based
Positiveory animals. The classical
Chemicals
nusson and Kligman, andof skin sensitisation. The
LLNA: DA (TG 442 A)
xclusively, and have also
terms of animal welfaren.

Soluble at 20 mg/ml

Negative

Insoluble at 20 mg/ml

Should not be
considered

Supposednegativessing the first key event(OECD TG 442D; AREed for contributing to the

Acceptance criteria

33.
The following acceptance criteria should be met when using the IL-8 Luc assay:n of cell surface marker(s)- Ind-IL8LA
posure to sensitisers(e.g. should be more than 5.0 at least in one concentration of the positive control, 4-NBB, in eachrun.
e activation of DC. Skiners such as CD40,
CD54, should be less than 1.4 at any concentration of the negative control, lactic acid, in each run.
- Ind-IL8LA
such as IL-1β -and
TNF-α,
Data from plates for which the GAPLA of control wells with cells and Tripluc but without chemicals is12), associatedlesswith
thanDC
5 times of that of well containing test medium only (50 µL/well of RPMI-1640 containing 10%
FBS and 50 µL/well of X-VIVOTM 15) should be rejected.
- Datansitisation AOP(2) from(13), plates for which the Inh-GAPLA of all concentrations of the test or control chemicals is lessthan
0.05 should be rejected. In this case, the first test should be repeated so the highest final concentration
one may not be
sufficientoftheemicals. Thereforerepeateddata test is the lowest final concentration of the previous test.
support the discriminationn used within Integrated
TEST REPORT
mplementary information,
tion AOP as well as non34. of data
The test report should include the following information:
ples of the use
ardised both in relation toive predictions, have been
Test chemicals- Mono-constituent substance:, neither to sub-categorise
authorities implementing
 Chemical identification, such as IUPAC or CAS name(s), CAS number(s), SMILES or InChI
ent decisions. However,methods may be used on code, structural formula, and/or other identifiers;
 Physical appearance, water solubility, molecular weight, and additional relevant
physicochemical properties, to the extent available;

Purity, chemical identity of impurities as appropriate and practically feasible, etc;

© OECD 2018
© OECD 2018

49

2 ││
50

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used topriorreadouts
to testing,
if applicable(e.g.bewarming,
grinding);
generate the Treatment
data and themeasuredbut canused indiscriminately
to address countries’
TM
TMthe AOP for skinrequirements
for test resultson the15.
Key
on activationof dendritic
cells of
ForEventchemicalsthat are insolublein X-VIVO
15, whether
 Solubility
in X-VIVO
sensitisation precipitation
while benefitingfrom theareMutual
Acceptanceof Data.
or flotation
observed
after centrifugation;Concentration(s) tested;

Backgroundand principlesof thetotest
included in the Key Event based Storage conditions
and stability
themethods
extent available;
Test GuidelineTM Justification for choice of solvent/vehicle for each test chemical if X-VIVO 15 has not been
4. The assessmentused. of skin sensitisation has typically involved the use of laboratory animals. The classicalmethods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The-murineMulti-constituent
substance,
UVCB
and its
mixture:tests, the LLNA(TG 429)(3) and
two non-radioactive modifications, LLNA: DA (TG 442 A)(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction response exclusively, and have also
 Characterisation
far as possible
by e.g.overchemicalidentity
above),
purity,quantitative
gained acceptance,since theyasprovide
an advantage
the guinea
pig(seetests
in termsof animal
welfareand
relevant physicochemical
propertiesof the constituents, to thetogether withoccurrence
an objective
measurementof the induction
phase of(seeskinabove)
sensitisation.
extent available;
5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event
 Physical appearance, water solubility, and additional relevant physicochemical properties, to(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; AREthe extent available;
Nrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to the ofMolecularweight or apparent
molecular
in case of mixtures/polymers of known
evaluation
the skin sensitisation
hazard potential
ofweight
chemicals.
compositions or other information relevant relevant for the conduct of the study;
6. Test methods described in this TG either quantify the change in the expression of cell surface marker(s) withTreatment
prior toif applicable(e.g. warming,
grinding); exposure to sensitisers (e.g.
associatedthe processof testing,activationof monocytes
and DC following
TM
TM
CD54, CD86)
or the changes
in IL-8expression,a cytokine
withthe activationof DC. Skin
 Solubility
in X-VIVO
15.
For chemicals
that areassociatedinsoluble in
X-VIVO15, whether
sensitisers havebeen reported
to inducethe expressionof cell membrane markers such as CD40, CD54,
precipitation
or flotation
are observed
after centrifugation;
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,
 Concentration(s)tested; IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
and several
chemokines including
activation
 (2).Storage conditions and stability to the extent available.
TM

has (2)
not (13),
 Justification
for choiceof solvent/vehicle
for eachtestofchemical,
X-VIVO 15
7. However,as DC activation
represents
only one key
event
the skin ifsensitisation
AOP
been used.with test methods measuring markers of DC activation alone may not be sufficient
information generated
to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore data
generated with the test methods described in this Test Guideline are proposed to support the discrimination
Controls
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
to Testing and Assessment (IATA), together with other relevant complementary information,-Approaches
Positive control:e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as nontesting methods,
includingread-acrosschemicalanalogues
the useor ofdata Chemical
identification,
suchfrom
as IUPAC
or CAS
name(s),(13).
CAS Examples
number(s),of
SMILES
InChI
generated withthese
methodsformula,within Defined
Approaches,
i.e. approaches standardised both in relation tocode,
structural
and/or otheridentifiers;
the set of information sources used and in the procedure applied to the data to derive predictions, have been (13)
Physicalwatersolubility,
molecular
published
and canappearance,
be employed
as usefulelements
withinweight,
IATA. and additional relevant
physicochemical properties, to the extent available and where applicable;
8. The test
methods
describedidentity
in this Test
Guidelineascannot
be usedtheir own,feasible,
neither to Purity, chemicalof impurities
appropriate
andonpracticallyetc;sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing optional
Treatment
prior to testing,applicablewarming,
grinding);
these twosubcategories,
norif topredict (e.g.
potency
for safetyassessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on Concentration(s) tested;
their own to classify a chemical into UN GHS category 1.
 Storage conditions and stability to the extent available;

Reference to historical positive control results demonstrating suitable acceptance criteria, if
applicable.
© OECD 2018
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OECD/OCDE

442E │ 51

o the procedure used to
y to address countries’- for
Negative
ls of the AOP
skin control:y Event based

Chemical identification, such as IUPAC or CAS name(s), CAS number(s), and/or otheridentifiers;Purity, chemical identity of impurities as appropriate and practically feasible, etc;

 Physical appearance, molecular weight, and additional relevant physicochemical properties inory animals. The classical the case other negative controls than those mentioned in the Test Guideline are used and to thenusson and Kligman, and extent available;of skin sensitisation. The
LLNA: DA (TG 442A) Storage conditions and stability to the extent available;
xclusively, and have also Justification for choice of solvent for each test chemical.
terms of animal welfaren.
Test method conditions
ssing the first key event- Nameand address of the sponsor, test facility and study director;(OECD TG 442D;
AREed for contributing
to the of test method used;- Description- Cell line used, its storage conditions, and source (e.g. the facility from which it was obtained);n of cell surface marker(s)- Lot number and origin of FBC, supplier name, lot number of 96-well flat-bottom black plate, and lot
posure to sensitisers (e.g.
number of Tripluc reagent;e activation of DC. Skiners such as CD40,
CD54,number and cell density used for testing;- Passage
such as IL-1β and TNF-α,- withCell counting
method used for seeding prior to testing and measures taken to ensure homogeneous cell12), associatedDC
number distribution;- Luminometer used (e.g. model), including instrument settings, luciferase substrate used, andnsitisation AOP (2)(13),
demonstrationof appropriate luminescence measurements based on the control test described in
one may not be sufficient
Appendix II;
emicals. Therefore data
- The procedure used to demonstrate proficiency of the laboratory in performing the test method (e.g. by
support the discrimination
testing of proficiency substances) or to demonstrate reproducible performance of the test method overn used within Integrated
time.
mplementary information,
tion AOP as well as nonples of the use
data
Test ofprocedure
ardised both in relation toive predictions,
beenof replicates and runs performed;- haveNumber- Test chemical concentrations, application procedure and exposure time (if different from thoserecommended);, neither to sub-categorise
authorities implementing- Description of evaluation and decision criteria used;
ent decisions. However,methods may -be Description
used on of study acceptance criteria used;- Description of any modifications of the test procedure.
Results© OECD 2018
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51

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52

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442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used to-generate
Measurements
IL8LA
and GAPLA;
the data ofandthe readouts
measured but can be used indiscriminately to address countries’
forfortest
resultsInd-IL8LA,
on the Key
on activation of dendritic cells of the AOP for skin-requirements
Calculations
nIL8LA,andEvent
Inh-GAPLA;
sensitisation while benefiting from the Mutual Acceptance of Data.
- The 95% confidence interval of Ind-IL8LA;- A graph depicting dose-response curves for induction of luciferase activity and viability;

Background and principles of the test methods included in the Key Event based- Descriptionof any other relevant observations, if applicable.
Test Guideline

4. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classicalDiscussion
ofuse
the guinea-pigs,
results
methods that
the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The-murineDiscussionof theresults
Luc assay;
tests, the
LLNA(TGobtained429) (3)withandtheits IL-8
two non-radioactive
modifications, LLNA: DA (TG 442 A)
(5)
and
LLNA:
BrdU-ELISA
(TG
442
B)
(6),
all
assesstheinductionandhave also- Consideration of the assay results in the context of an IATA, if otherresponse
relevant exclusively,
information isavailable.
gained acceptance, since they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
Conclusion
5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; ARENrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals.
6. Test methods described in this TG either quantify the change in the expression of cell surface marker(s)associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
sensitisers have been reported to induce the expression of cell membrane markers such as CD40, CD54,
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
activation (2).
7. However, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficient
to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore data
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as nontesting methods, including read-across from chemical analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

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OECD/OCDE

442E │ 53

o the procedure used to
y to address LITERATURE
countries’
ls of the AOP for skin
1.
Takahashi T, Kimura Y, Saito R, Nakajima Y, Ohmiya Y, Yamasaki K, and Aiba S. 2011. An in
vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8. Toxicol
Sci 124:359-69.

y Event based2.
OECD (2017), Validation report for the international validation study on the IL-8 Luc assay as atest evaluating the skin sensitizing potential of chemicals conducted by the IL-8 Luc Assay. Series on
ory animals. The
classical
Testingand Assessment No. 267, ENV/JM/MONO(2017)19. Organisation for Economic Cooperation andnusson and Kligman,
andDevelopment,
Paris. Available at: [http://www.oecd.org/env/ehs/testing/series-testing-assessmentof skin sensitisation.
Thepublications-number.htm].
LLNA: DA (TG 442 A)
xclusively, and3. have also
OECD (2017), Report of the Peer Review Panel for the IL-8 Luciferase (IL-8 Luc) Assay for interms of animal
vitrowelfare
skin sensitisation. Series on Testing and Assessment No. 258, ENV/JM/MONO(2017)20.
n.
Organisationfor
Economic
Cooperation
andDevelopment,
Paris.
Availableat:[http://www.oecd.org/env/ehs/testing/series-testing-assessment-publications-number.htm].
ssing the first key event(OECD TG 442D;
ARE2.
OECD (2016), Guidance Document On The Reporting Of Defined Approaches And Individualed for contributing
to the Sources To Be Used Within Integrated Approaches To Testing And Assessment (IATA) For
Information
Skin Sensitisation, Series on Testing & Assessment No. 256, ENV/JM/MONO(2016)29. Organisation for
Economic Cooperation and Development, Paris. Available at: [http://www.oecd.org/env/ehs/testing/seriesn of cell surface
marker(s)testing-assessment-publications-number.htm].
posure to sensitisers (e.g.
e activation of
3. DC. Skinvan der Veen JW, Rorije E, Emter R, Natsch A, van Loveren H, and Ezendam J. 2014.
ers such as CD40,
CD54, the performance of integrated approaches for hazard identification of skin sensitizing
Evaluatingsuch as IL-1β chemicals.
and TNF-α,Regul Toxicol Pharmacol 69:371-9.
12), associated with DC
4.
Kimura Y, Fujimura C, Ito Y, Takahashi T, Nakajima Y, Ohmiya Y, and Aiba S. 2015.
Optimization of the IL-8 Luc assay as an in vitro test for skin sensitization. Toxicol In Vitro 29:1816-30.
nsitisation AOP (2) (13),
5. sufficient
Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle S, Landsiedel R, Jaworska J, Kern
one may not be
PS, Gerberick
emicals. Therefore
data F, et al. 2015. Assessing skin sensitization hazard in mice and men using non-animal test
methods. Regul Toxicol Pharmacol 71:337-51.
support the discriminationn used within Integrated
6.
Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y,
mplementary information,
Nishiyamation AOP as well
as non-N, and Itagaki H. 2010. A comparative evaluation of in vitro skin sensitisation tests: the humancell-line
activation test (h-CLAT) versus the local lymph node assay (LLNA). Alternatives to laboratory
ples of the useof dataanimals:
38:275-84.
ardised both in relation ATLAtoive predictions, have been
7.
Patlewicz G, Casati S, Basketter DA, Asturiol D, Roberts DW, Lepoittevin J-P, Worth A and
Aschberger K (2016) Can currently available non-animal methods detect pre and pro haptens relevant forskin sensitisation? Regul Toxicol Pharmacol, online.
, neither to sub-categorise
authorities implementing
Thorne N, Inglese J, and Auld DS. 2010. Illuminating insights into firefly luciferase and otherent decisions.8. However,bioluminescentreporters used in chemical biology. Chem Biol 17:646-57.
methods may be used on
9.
OECD (2016), Test No. 455: Performance-Based Test Guideline for Stably TransfectedTransactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists, OECD Publishing,Paris. http://dx.doi.org/10.1787/9789264265295-en.

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53

2 ││
54

442E
442E

OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used to10.
Viviani
UchidaA, Suenaga
N, Ryufuku
M, beandusedOhmiyaY. 2001. Thr226
is a key countries’
residue forgenerate thedataV,
andthe readouts
measuredbut canindiscriminately
to address
bioluminescence
determination
in beetle
luciferases.
Biochem
Biophys cells
Res Commun
280:1286-91.
requirements for spectra
test resultson the Key
Event
on activationof dendriticof the AOP
for skinsensitisation while benefiting from the Mutual Acceptance of Data.
11.
Viviani VR, Bechara EJ, and Ohmiya Y. 1999. Cloning, sequence analysis, and expression ofactive Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primarystructures.
Biochemistry38:8271-9.
Backgroundandprinciples of the test methods included in the Key Event based

Test Guideline

12.
Nakajima Y, Kimura T, Sugata K, Enomoto T, Asakawa A, Kubota H, Ikeda M, and Ohmiya Y.
4. TheMulticolor
assessmentluciferaseof skin sensitisation
hasone-step
typicallymonitoring
involved theof laboratory
animals. The
2005.
assay system:of usemultiplegene expressions
with classicala single
methods thatuse guinea-pigs,
the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andsubstrate.
Biotechniques
38:891-4.
the Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The
13.
OECD
To be
published
PerformanceStandards modifications,for the assessmentof proposed
similarmurine tests,
the (2017),
LLNA (TG429)(3) and- its
two non-radioactive
LLNA:
DA (TG 442
A)orin BrdU-ELISA
vitro skin sensitisation
test methods.
OECDresponse
Environment,
HealthSafety(5) modified
and LLNA:(TG 442 B)IL-8(6), luc
all assess
the induction
exclusively,
andandhave
also
Publications,
Series sinceon Testingand Assessment.
OECD,
Paris,
gained acceptance,
they provide
an advantage
overthe France.
guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
14.
OECD (2005), Guidance Document the Validation and International Acceptance of New or
Updated
Testmechanistically-based
Methods for HazardinAssessment.
Environment,
and Safety
publications,
5. Recently
chemico andOECD
in vitro
test methodsHealthaddressing
the first
key eventOECD
on Testing
Assessment
No.34.
OECD,
France.key event (OECD TG 442D; ARE(OECDSeries
TG 442C;
Directand
PeptideReactivity
Assay(7)),Paris,and second
Nrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to the15.
(2017),
Draft Guidance
document:
In Vitro Method Practices (GIVIMP) for theevaluationOECD
of the skinsensitisation
hazard potential
of Goodchemicals.
Development and Implementation of In Vitro Methods for Regulatory Use in Human Safety Assessment.
Organisationfor
Economic
Cooperation
andDevelopment,
Available
at:
6. Test methods describedin this TG either
quantify thechangein the expressionParis.
of cell surface
marker(s)[http://www.oecd.org/env/ehs/testing/OECD%20Draft%20GIVIMP_v05%20-%20clean.pdf].
associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
16.
(2016),
Luc of cell
assay
protocol,at.
sensitisersJaCVAM
have been reported
to induceIL-8
the expression
membranemarkers suchAvailable
as CD40, CD54,
http://www.jacvam.jp/en_effort/effort02.html.
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
17.
activationNiwa(2). K, Ichino Y, Kumata S, Nakajima Y, Hiraishi Y, Kato D, Viviani VR, and Ohmiya Y.
2010. Quantum yields and kinetics of the firefly bioluminescence reaction of beetle luciferases. Photochem
Photobiol
86:1046-9.
7. However,as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficient18.
OECD(2012),The orAdverse
Outcomefor SkinSensitisation
InitiatedTherefore
by Covalent
to conclude
on thepresenceabsenceof skin Pathway
sensitisation
potentialof chemicals.
data
Binding
toProteins,Part
1:
Scientific
Evidence.
OECD
Environment,
Healthand
SafetyPublications,generated with the test methods described in this Test Guideline are proposed to support the discrimination
Serieson skinTestingand Assessment
168.Category
OECD, Paris,
France.
betweensensitisers(i.e. UNNo.
GHS
1) andnon-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,
19.
UnitedNations(2015),
GloballyotherHarmonizedof sensitisation
ClassificationAOP
andasLabellingofe.g. derived
from invitro assays
addressing
key eventsSystemof the skinwell as nonChemicals(GHS).including
Sixth revised
edition. fromNew York& Geneva:
UnitedNations
Publications.
ISBN:
testing methods,
read-acrosschemicalanalogues(13).
Examplesof the useof 978data
92-1-117006-1.
Available
at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html.
generated with thesemethods within
Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

© OECD 2018
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54

OECD/OCDEo the procedure used to
Iy to address APPENDIX
countries’
ls of the AOP for skin

442E │ 55

DEFINITIONSy Event basedAccuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
with concordance to mean the proportion of correct outcomes of a test method (16).
ory animals. The classicalnusson and Kligman,andAOP (Adverse
Outcome Pathway): Sequence of events from the chemical structure of a target chemicalof skin sensitisation.
Theor group of similar chemicals through the molecular initiating event to an in vivo outcome of interest (20).
LLNA: DA (TG 442 A)
xclusively, andhave Cellalso viability 05. Minimum concentration at which chemicals show less than 0.05 of Inh-GAPLA.
CV05:
terms of animal welfaren.
FInSLO-LA: Abbreviation used in the validation report and in previous publications regarding the IL-8

Luc assay to refer to Ind-IL8LA. See Ind-IL8LA for definition.
ssing the first key event(OECD TG 442D; AREGAPLA: Luciferase Activity of Stable Luciferase Red (SLR) (max = 630 nm), regulated by GAPDH
ed for contributing to thepromoter and demonstrates cell viability and viable cell number.

Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
n of cell surface marker(s)organism, system or (sub) population is exposed to that agent.
posure to sensitisers (e.g.
e activation of DC. Skin
IATA (Integrated Approach to Testing and Assessment): A structured approach used for hazard
ers such as CD40, CD54,
identification (potential), hazard characterisation (potency) and/or safety assessment (potential/potency
such as IL-1β and TNF-α,and exposure) of a chemical or group of chemicals, which strategically integrates and weights all relevant12), associated with DC
data to inform regulatory decision regarding potential hazard and/or risk and/or the need for further
targeted and therefore minimal testing.

nsitisation AOP (2) (13),II-SLR-LA:
one may not be
sufficient Abbreviation used in the validation report and in previous publications regarding the IL-8
Luc
assay
to refer to Inh-GAPLA. See Inh-GAPLA for definition
emicals. Therefore
data
support the discrimination(Interleukin-8): A cytokine derived from endothelial cells, fibroblasts, keratinocytes, macrophages,
n used withinIL-8
Integrated
andmonocytes
mplementary information, that causes chemotaxis of neutrophils and T-cell lymphocytes.
tion AOP as well as nonIL8LA:
Luciferase Activity of Stable Luciferase Orange (SLO) (max = 580 nm), regulated by IL-8
ples of the useof dataardised both inpromoter.
relation toive predictions, have been
Ind-IL8LA: Fold induction of IL8LA. It is obtained by dividing the nIL8LA of THP-G8 cells treated withchemicals by that of non-stimulated THP-G8 cells and represents the induction of IL-8 promoter activity
by chemicals.
, neither to sub-categorise
authorities implementing
ent decisions.Inh-GAPLA:
However, Inhibition of GAPLA. It is obtained by dividing GAPLA of THP-G8 treated with chemicals
with
GAPLA
methods may be used
on of non-treated THP-G8 and represents cytotoxicity of chemicals.
Minimum induction threshold (MIT): the lowest concentration at which a chemical satisfies the positive
criteria
Mixture: A mixture or a solution composed of two or more substances in which they do not react.
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OECD/OCDE
OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used togenerate the data and the readouts measured but can be used indiscriminately to address countries’
Mono-constituent
substance:
A substance,
definedits quantitative
composition,requirements for test
results on
the Key Eventon byactivationof dendritic
cells of inthewhich
AOPoneformainskin
constituent
ispresent
to
at
least
80%
(w/w).
sensitisation while benefiting from the Mutual Acceptance of Data.
Multi-constituent substance: A substance, defined by its quantitative composition, in which more than
one of Backgroundthe main constituentsis present
in atest
concentration≥ 10% (w/w)
80% based(w/w). A multiand principlesof themethods includedin the andKey <Event
constituent
substanceistheresultofamanufacturing
process.
The
difference
betweenmixtureand multiTest Guideline
constituent substance is that a mixture is obtained by blending of two or more substances without chemical
4. The assessmentof skin sensitisationhas
thereaction.
use of laboratory animals. The classicalreaction.
A multi-constituent
substance isthetypicallyresult ofinvolveda chemical
methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler
Test(TGluciferase
406) (4),activity
assess boththe inductionand elicitation
phases ofnormalised
skin sensitisation.
The
nIL8LA:
The
SLO
reflecting
IL-8 promoter
activity (IL8LA)
by the SLR
murine tests,
the LLNA(TGGAPDH
429) (3)promoter
and its two
non-radioactive
LLNA:
DA activity(TG 442afterA)
luciferase
activityreflecting
activity(GALPA). Itmodifications,represents IL-8
promoter(5) and LLNA:(TG
442 B) (6), all assess the induction response exclusively, and have also
consideringcell BrdU-ELISA
viability or cellnumber.
gained acceptance, since they provide an advantage over the guinea pig tests in terms of animal welfare
together withAbbreviation
an objective usedmeasurementof the induction
phaseof skin sensitisation.
nSLO-LA:in the validation
report andin previous
publications regarding the IL-8 Luc
assay to refer to nIL8LA. See nIL8LA for definition
5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event(OECD TG
442C;ADirect
PeptideReactivity
Assay (7)), andkey event(OECDwith
TG a442D;
AREPositive
control:
replicatecontaining
all components
of asecond
test systemand treatedsubstance
Nrf2 Luciferase
Methodresponse.
(8)) of thesensitisation
AOP havebeen
adoptedfor contributing
to theknown
to induceTest
a positive
Toskinensurethat variability
in thepositive
control
response acrossevaluation
of
the
skin
sensitisation
hazardpotentialofchemicals.
time can be assessed, the magnitude of the positive response should not be excessive.
6. Test methods
describedwhichin thisbecome
TG eitherquantifythroughthe change
in transformation.
the expression of cell surface marker(s)Pre-haptens:
Chemicalssensitisers
abiotic
associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) Chemicals
or the changes
in IL-8expression,a cytokine
with thepotential.
activation of DC. Skin
Pro-haptens:
requiring
enzymatic
activation
to exertassociatedskin sensitisation
sensitisers have been reported to induce the expression of cell membrane markers such as CD40, CD54,
CD80, CD83,
and CD86 ofin relationshipaddition to induction
cytokines,
such as IL-1β
and TNF-α,
Relevance:
Descriptionof the testoftoproinflammatory
the effect of interest
and whether
it is meaningful
andseveralchemokines
including
IL-8(CXCL8)
and
CCL3
(9)
(10)
(11)
(12),
associatedwith theDCand useful for a particular purpose. It is the extent to which the test correctly measures or predicts
activation(2).
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (16).
7. However, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated
withmethods
DC activation
alone maynotandbe between
sufficient
Reliability:
Measures ofthetestextentthat a measuring
test methodmarkers
can be of
performed
reproducibly
withinto conclude overon thepresenceor absenceof skinsensitisationof chemicals.
Therefore
data
laboratories
time,when performed
using
the same
protocol.potential
It is assessedby calculating
intra- andgenerated
with
the
test
methods
described
in
this
Test
Guideline
areproposedtosupportthediscriminationinter-laboratory reproducibility and intra-laboratory repeatability (16).
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches
Testing
togetherwith other relevant
information,
Run:
A run toconsistsof andone Assessment
or more test (IATA),
chemicals
tested concurrently
with a complementary
solvent/vehicle control
ande.g.
derived
from
in
vitro
assaysaddressing
other
key
eventsoftheskinsensitisation
AOP
as
well
as nonwith a positive control.
testing methods, including read-across from chemical analogues (13). Examples of the use of data
generated withmethods
Defined Approaches,standardisedboth
relation
Sensitivity:
Thethese
proportionof within
all positive/active
chemicals i.e.
thatapproaches
are correctlyclassified bytheintest.
It is ato
the
set
of
information
sources
used
and
in
the
procedureapplied
to
the
data
to
derivepredictions,
have
been
measure of accuracy for a test method that produces categorical results, and is an important consideration
published
(13)
and
can
be
employedasusefulelements
withinIATA.
in assessing the relevance of a test method (16).
8. The test Abbreviation
methods describedin thethisvalidation
Test Guideline
used onpublications
their own, neither
to sub-categorise
SLO-LA:
used inreportcannot
and inbeprevious
regarding
the IL-8 Luc
skinsensitisers
into
subcategories
1A
and
1B
as
defined
by
UN
GHS
(1),
for
authorities
implementing
assay to refer to IL8LA. See IL8LA for definition.
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending Abbreviationon the regulatory
positive
results
generatedthese methods
maythebeIL-8usedLucon
SLR-LA:
used framework,in the validation
reportand inpreviouswith
publications
regarding
their
own
to
classifyachemical
into
UN
GHS
category
1.
assay to refer to GAPLA. See GAPLA for definition.
Solvent/vehicle control: An untreated sample containing all components of a test system except of thetest chemical, but including the solvent/vehicle that is used. It is used to establish the baseline response© OECD 2018
© OECD 201856

OECD/OCDE

442E │ 57

o the procedure used tothe samples treated with the test chemical dissolved or stably dispersed in the same solvent/vehicle.
y to address forcountries’
Whentested with a concurrent medium control, this sample also demonstrates whether the solvent/vehicle
ls of the AOP
for skininteracts with the test system.

Specificity: The proportion of all negative/inactive chemicals that are correctly classified by the test. It is ay Event basedmeasure of accuracy for a test method that produces categorical results and is an important consideration inassessing the relevance of a test method (16).

ory animals. The
classical Chemical elements and their compounds in the natural state or obtained by any production
Substance:
nusson and Kligman,process, andinducing any additive necessary to preserve the stability of the product and any impurities
of skin sensitisation.
derivingThefrom the process used, but excluding any solvent which may be separated without affecting the
LLNA: DA (TG
442 ofA)the substance or changing it composition.
stability
xclusively, and have also
terms of animal
welfare Also called surface-active agent, this is a substance, such as a detergent, that can reduce theSurfactant:n.
surface tension of a liquid and thus allow it to foam or penetrate solids; it is also known as a wetting agent.
(TG437)ssing the first key event(OECD TG 442D;
ARETest chemical:
The term "test chemical" is used to refer to what is being tested.
ed for contributing to the
THP-G8: An IL-8 reporter cell line used in IL-8 Luc assay. The human macrophage-like cell line THP-1
was transfected the SLO and SLR luciferase genes under the control of the IL-8 and GAPDH promoters,n of cell surface
marker(s)respectively.
posure to sensitisers (e.g.
e activation of
DC. Skin
UnitedNations Globally Harmonized System of Classification and Labeling of Chemicals (UNers such as CD40,
CD54,
GHS):
A system proposing the classification of chemicals (substances and mixtures) according tosuch as IL-1β standardised
and TNF-α, types and levels of physical, health and environmental hazards, and addressing12), associatedwith DCcorresponding
communication elements, such as pictograms, signal words, hazard statements,
precautionary statements and safety data sheets, so that to convey information on their adverse effects
with a view to protect people (including employers, workers, transporters, consumers and emergency
nsitisation AOP(2) (13), and the environment (21).
responders)
one may not be sufficientemicals. Therefore
UVCB:data
substances of unknown or variable composition, complex reaction products or biologicalsupport the discriminationmaterials.
n used within Integrated
mplementary information,
Valid test method: A test method considered to have sufficient relevance and reliability for a specifiction AOP as well
as nonpurpose
and which is based on scientifically sound principles. A test method is never valid in an absolute
ples of the useof
data
sense, but
only in relation to a defined purpose.
ardised both in relation toive predictions, have been

, neither to sub-categorise
authorities implementing
ent decisions. However,methods may be used on© OECD 2018
© OECD 2018

57

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2 │58 │

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OECD/OCDE

3. The test methods included in this Test Guideline may differ in relation to the procedure used togenerate the data and the readouts measured but can be used indiscriminately to address countries’
APPENDIX
requirements
for IItest results on the Key Event on activation of dendritic cells of the AOP for skinsensitisation while benefiting from the Mutual Acceptance of Data.
PRINCIPLE OF MEASUREMENT OF LUCIFERASE ACTIVITY AND DETERMINATION
OF THE TRANSMISSION COEFFICIENTS OF OPTICAL FILTER FOR SLO AND SLR

Background and principles of the test methods included in the Key Event based
Test Guideline

MultiReporter
System
-Triplucbe usedinvolved
with a microplate-type
luminometerwithmulti4. The
assessmentAssayof skinsensitisation
hascantypically
the use of laboratory
animals.
Thea classicalcolourdetectionsystem,
which
can
equip
an
opticalfilter(e.g.
Phelios
AB-2350(ATTO),
ARVO
methods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andTristar
LB941(Berthold)).
Theinduction
optical filter
in measurement
is 600–620
nm longthe(PerkinElmer),
Buehler Test (TG406)(4), assess
both theand usedelicitation
phases of skinsensitisation.
The
or
short
pass
filter,or
600–700
nm
band
pass
filter.
murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction response exclusively, and have also
(1) Measurementof two-colour
luciferases
with an overopticalgainedacceptance, since
they provide
an advantage
thefilter.
guinea pig tests in terms of animal welfare
This
is
an
exampleusing
Phelios
AB-2350(ATTO).
This
luminometeris equipped with a 600 nm
together with an objective measurement of the induction phase ofskin sensitisation.
long pass filter (R60 HOYA Co.), 600 nm LP, Filter 1) for splitting SLO (max = 580 nm) and SLR
(max
= 630
nm) luminescence. in chemico and in vitro test methods addressing the first key event
5. Recently
mechanistically-based(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; AREToLuciferase
determineTesttransmission
coefficientsthe 600 nm
LP,havefirst,
using
purified
SLO and SLR
Nrf2
Method (8))
of the skinofsensitisation
AOP
been
adoptedfor contributing
to theluciferase
enzymes,measurei)theintensity
of
SLO
and
SLR
bioluminescenceintensity
without filter
evaluation of the skin sensitisation hazard potential of chemicals.
(F0), ii) the SLO and SLR bioluminescence intensity that passed through 600 nm LP (Filter 1), andiii) calculatetransmissionof 600 nm
for SLO
andexpression
SLR listedofbelow.
6. Testmethodsthe
described
in thiscoefficients
TG either quantify
theLPchangein thecell surface marker(s)associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
coefficients
Abbreviation
CD54,TransmissionCD86) or thechanges in IL-8
expression,Definitiona cytokine associated with the activation of DC. Skin
Filter1
sensitisers have beenreported
to induce the expressionof cell membrane markers such as CD40, CD54,
The filter’s transmission
�OtoR60induction of
SLOCD83, and CD86
Transmission
CD80,
in addition
proinflammatory
cytokines, such as IL-1β and TNF-α,coefficient for the SLO
coefficientsand several chemokines
including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
Filter 1
activation (2).
The filter’s transmission
SLR
Transmission �R R60
coefficient for the SLR
coefficients
7. However, as DC
activation represents only one key event of the skin sensitisation AOP (2) (13),information
test methods
markers
DC activation
alone
may not be sufficient
When thegenerated
intensity withof SLOand SLRmeasuring
in test sample
areofdefinedas O andR, respectively,i) theto intensity
concludeofonlightthe without
presenceorabsenceofskinsensitisation
potentialofchemicals.
Therefore
data
filter (all optical) F0 and ii) the intensity of light that transmits through 600
generated
with
the
test
methods
described
in
this
Test
Guideline
areproposedtosupportthediscrimination
nm LP (Filter 1) F1 are described as below.
between skinF0=O+R
sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,x Oassays+ RR60xR
e.g. derived F1=O
from inR60
vitro
addressing
other key events of the skin sensitisation AOP as well as nontesting
methods,
includingread-acrossfrom
These formulas can be rephrased as follows: chemical analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to1
1 usedO and in the procedure applied to the data to derive predictions, have been
F0of information
the setsources) ( )as useful elements within IATA.
( ) =(13)( and can be employedpublishedκ𝑅𝑅𝑅𝑅60κ𝑂𝑂𝑅𝑅60
R
F1
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categorise
Then using calculated transmittance factors (OR60 and RR60) and measured F0 and F1, you canskin sensitisers into subcategories 1A and 1B as definedby UN GHS (1), for authorities implementing
calculate O and R-value as follows:these two optional subcategories,
nor
topredict
potency
for safety assessment decisions. However,
−1
1
1
O
F0
depending on the regulatory framework, positive results generated with these methods may be used on( own) =to(classify a chemical) into( UN) GHS category 1.
their
κ𝑅𝑅𝑅𝑅60κ𝑂𝑂𝑅𝑅60
R
F1
Materials and methods for determining transmittance factor© OECD 2018
© OECD 201858

OECD/OCDE

442E │ 59

o the procedure used to(1) Reagentsy to address countries’∙ Singlels of the AOP forskin purified luciferase enzymes:
Lyophilised purified SLO enzyme
Lyophilised purified SLR enzyme(which for the validation work were obtained from GPC Lab. Co. Ltd., Tottori, Japan with THP-G8
y Event based cell line)∙ Assay reagent:
Tripluc® Luciferase assay reagent ( for example from TOYOBO Cat#MRA-301)
ory animals. The ∙classicalMedium: for luciferase assay (30 ml, stored at 2 – 8°C)
nusson and Kligman, andof skin sensitisation. The
Final conc. in Required
LLNA: DA (TG 442 A)
ReagentConc.
xclusively, and have also
mediumamountterms of animal welfare
RPMI-1640
27 ml
n.

FBS
ssing the first key event(OECD TG 442D; AREed for contributing
the(2) toPreparationof enzyme solution

-

10 %

3 ml

Dissolve lyophilised purified luciferase enzyme in tube by adding 200 μl of 10 ~ 100 mM Tris/HCl or
Hepes/HCl (pH 7.5 ~ 8.0) supplemented with 10% (w/v) glycerol, divide the enzyme solution into 10
n of cell surface marker(s)μl aliquots in 1.5 ml disposable tubes and store them in a freezer at -80°C. The frozen enzyme
posure to sensitisers(e.g.can be used for up to 6 months. When used, add 1 ml of medium for luciferase assay (RPMIsolution
e activation of DC.
1640Skinwith 10% FBS) to each tube containing the enzyme solutions (diluted enzyme solution) anders such as CD40,
CD54,
keep
them on ice to prevent deactivation.
such as IL-1β and TNF-α,12), associated withDC(3) Bioluminescencemeasurement

Thaw Tripluc® Luciferase assay reagent (Tripluc) and keep it at room temperature either in a water
bath or at ambient air temperature. Power on the luminometer 30 min before starting the measurement
nsitisation AOP (2) (13),to allow the photomultiplier to stabilise. Transfer 100 μl of the diluted enzyme solution to a black 96
one may not be sufficient
well plate (flat bottom) (the SLO reference sample to #B1, #B2, #B3, the SLR reference sample toemicals. Therefore data
#D1, #D2, #D3). Then, transfer 100 μl of pre-warmed Tripluc to each well of the plate containing the
support the discrimination
diluted enzyme solution using a pipetman. Shake the plate for 10 min at room temperature (about
n used within Integrated
25°C) using a plate shaker. Remove bubbles from the solutions in wells if they appear. Place the plate
mplementary information,in the luminometer to measure the luciferase activity. Bioluminescence is measured for 3 sec each intion AOP as well as nonthe absence (F0) and presence (F1) of the optical filter.
ples of the use of data
Transmission
coefficient of the optical filter was calculated as follows:
ardised both in relation
toTransmission
coefficient (SLO (OR60))= (#B1 of F1+ #B2 of F1+ #B3 of F1) / (#B1 of F0+ #B2 ofive predictions, havebeen
F0+ #B3 of F0)
Transmission coefficient (SLR (RR60))= (#D1 of F1+ #D2 of F1+ #D3 of F1) / (#D1 of F0+ #D2 of, neither to sub-categorise
F0+ #D3 of F0)
authorities implementing
Calculated transmittance factors are used for all the measurements executed using the sameent decisions. However,methods may beluminometer.
used onQuality control of equipment
The procedures described in the IL-8 Luc protocol should be used (18).
.
© OECD 2018
© OECD 2018

59

442E
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OECD/OCDE
OECD/OCDE│ │260

3. The test methods included in this Test Guideline may differ in relation to the procedure used togeneratethe data
APPENDIX
IIIand the readouts measured but can be used indiscriminately to address countries’
requirements for test results on the Key Event on activation of dendritic cells of the AOP for skinsensitisation while benefiting from the
Mutual Acceptanceof Data.
PROFICIENCYSUBSTANCES

Background and principles of the test methods included in the Key Event based

Prior to routine use of the test method described in this Annex to Test Guideline 442E, laboratories
Guideline
shouldTest
demonstratetechnical proficiency by obtaining the expected IL-8 Luc assay prediction for the 9
recommended
in Table 1 andby obtaining
valueswithin theanimals.
respective
4.substances
The assessmentof skin sensitisation
has typicallyinvolved
thethatuse fallof laboratory
Thereferenceclassical
range
for
at
least
8
out
of
the
9
proficiency
substances(selected
torepresent
the
range
of
responses
formethods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andskinsensitisation
hazards).
Other
selection
criteria
were andthat elicitation
the substances
areofcommerciallyavailable,
theBuehler
Test (TG406) (4),assessboth theinductionphasesskin sensitisation.
Theand thathigh-quality
vivo429)
referencedata
well
as high quality
in vitro data LLNA:
generated
IL-8
murinetests,
the LLNAin(TG(3) andits astwo
non-radioactive
modifications,
DAwith(TGthe
442
A)
Luc
assay
are available.
Also,(TGpublisheddata arethe IL-8exclusively,
Luc assay (6)
(5)
and
LLNA:
BrdU-ELISA
442 B)reference(6), all assesstheavailable
inductionfor
response
and(1).
have also
1: Recommended
demonstrating
technical
Luc welfare
assay
gainedTable
acceptance,since they substances
provide anfor
advantageover theguinea proficiency
pig tests in withtermstheofIL-8
animalSolubility
Reference range
together with an objective measurement of theinduction phase of skin sensitisation.
3in(μg/mL)XIL-8 Luc
In vivo2
5. Recently mechanistically-based in chemicoand inprediction
vitro 1test prediction
methods
addressing
the Luc
first key event
VIVO15
IL-8
CV054
5
at
20
(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECDMITTG 442D; AREmg/mL
Proficiency substances

CAS no.

State

Nrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theSensitiser
evaluationof the skin sensitisation
2,4-Dinitrochlorobenzene
97-00-7 hazard
Solid potential
Insoluble of chemicals. Positive
2.3-3.9
0.5-2.3(Extreme)

6. Test methods described in this TG either quantify thechange in the expression of cell surface marker(s)Sensitiser
Formaldehyde
Liquid9-30(Strong)
associatedwith the process50-00-0
of activationofSoluble
monocytes
and DC Positive
following exposure
to4-9sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
Sensitiser
sensitisers
have been reported
to induce
expressionof cell membranemarkers
as CD40, CD54,
2-Mercaptobenzothiazole
149-30-4
Solid theInsoluble
Positive250-290 such
60-250(Moderate)
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,and several chemokines including IL-8 (CXCL8) andCCL3 (9) (10) (11) (12), associated with DC
Sensitiser
Ethylenediamine
107-15-3
LiquidSolublePositive
500-700
0.1-0.4(Moderate)
activation (2).
Ethyleneglycol

Sensitiser

97-90-5
Liquid Insoluble
0.04-0.1
7. dimethacrylate
However, as DC activation
represents
only one key
event ofPositive
the skin >2000
sensitisation
AOP (2) (13),(Weak)
information generated with test methods measuring markers
ofDC
activation
alone
may
not
be sufficient
Sensitiser
4-Allylanisole(Estragol)
140-67-0
LiquidInsoluble
Positive>2000
0.01-0.07
to conclude on the presence or absence of skin sensitisation
potential of chemicals. Therefore data
(Weak)
generated with the test methods described in this Test Guideline
are proposed to support the discrimination
NonStreptomycin sulphate
3810-74-0
Solid
SolubleNegative
>2000
>2000
between skin sensitisers (i.e. UN GHS Category 1)sensitiser
and non-sensitisers when used within Integrated
NonNegative>2000 information,
GlycerolLiquidSolubletogether with otherApproaches
to Testing and 56-81-5
Assessment(IATA),
relevant >2000
complementarysensitisere.g. derived
from
in
vitro
assaysaddressing
other
key
eventsNon- of the skin sensitisation AOP as well as nonNegative
>2000
>2000
Isopropanol
67-63-0LiquidSolublesensitiser
testing methods, including read-across from chemical
analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
Abbreviations:
CAS sources
no. = Chemical
Service Registrytheset of information
used andAbstracts
in the procedure
applied toNumber
the data to derive predictions, have been1
published(13)potency
and canisbederived
employedas theusefulelements
withinThe in vivo
using
criteria
proposedbyIATA.
ECETOC (19).
2Based on historical observed values (1) (6).
8.3 The test methods described in this Test Guideline cannot be used on their own, neither
to sub-categorise
CV05 and IL-8 Luc MIT were calculated using water solubility given by EPI SuiteTM.
skinsensitisers
into
subcategories
1A
and
1B
as
defined
by
UN
GHS
(1),
for
authorities
implementing
4
CV05:
theoptional
minimum
concentrationnor
at whichchemicals
showforlesssafety
than 0.05
of Inh-GAPLA.
these
two
subcategories,
topredict
potencyassessment
decisions.
However,5MIT:thelowestconcentrations
at
which
a
chemicalsatisfies
thepositive
criteria.
depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

OECD2018
2018©©OECD60

OECD/OCDE

442E │ 61

o the procedure used to
y to address countries’
ls of the AOPAPPENDIX
for skinIVINDEXES AND JUDGMENT CRITERIAy Event based

ory animals. The
classical(nSLO-LA)
nIL8LAnusson and Kligman, andThe j-th repetition (j = 1-4) of the i-th concentration (i = 0-11) is measured for IL8LA (SLO-LA) andof skin sensitisation. The
GAPLA (SLR-LA) respectively. The normalised IL8LA, referred to as nIL8LA (nSLO-LA), and is
LLNA: DA (TG 442 A)
defined as:
xclusively, and have also
nIL8LA = IL8LA ij / GAPLA ij.
terms of animal welfare ij
This is the basic unit of measurement in this assay.
n.

(FInSLO-LA)
ssing the first key Ind-IL8LA
event
The
fold
increaseof the averaged nIL8LA (nSLO-LA) for the repetition on the i-th concentration(OECD TG 442D; AREcompareded for contributing
to thewith it at the 0 concentration, Ind-IL8LA, is the primary measure of this assay. This ratio iswritten by the following formula:







Ind-IL8LAi = 1 / 4  jnIL8LA ij / 1 / 4  jnIL8LA 0 j .
n of cell surface marker(s)posure to sensitisers (e.g.
e activation ofThe
DC.leadSkinlaboratory has proposed that a value of 1.4 corresponds to a positive result for the tested
ers such as CD40,
CD54,
chemical. This value is based on the investigation of the historical data of the lead laboratory. Datasuch as IL-1β and
TNF-α, team then used this value through all the phases of validation study. The primary outcome,management12), associatedInd-IL8LA,
with DC is the ratio of 2 arithmetic means as shown in equation.



95% confidence interval (95% CI)
nsitisation AOP (2) (13),The 95% confidence interval (95% CI) based on the ratio can be estimated to show the precision of
one may not be sufficient
this primary outcome measure. The lower limit of the 95% CI  1 indicates that the nIL8LA with the i-th
emicals. Therefore data
concentration is significantly greater than that with solvent control. There are several ways to constructsupport the discrimination
the 95% CI. We used the method known as Fieller’s theorem in this study. This 95% confidence interval
n used within Integrated
theorem is obtained from the following formula:
mplementary information,
tion AOP as well as non  B  B 2  4AC  B  B 2  4AC ples of the use of data,,ardised both in relation to
2A
2A



ive predictions, have been
A x 02  t 20.975  
where, neither to sub-categorise
authorities implementing
ent decisions. x
However,= 1 / n  methods may be 0used on0

sd2yi
sd02
2
2
B 2  x 
y , C y i  t 0.975   ,, and n 0 =4,n0n yi, sd 1 / n  1  nIL8LA  x  , nIL8LA , sd 1/n  1  nIL8LA  y  .
n
=4, y 1 / n   nIL8LA
yi

i

0j

j

yi

j

2

2
0

0

ij

2
yi

j

0j

0

2

yj

j

ij

i

t 0.975  is 97.5 percentile of the central t distribution with the  of the degree of freedom, where©©OECD
OECD2018
2018

61

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OECD/OCDE

│ │
262

2
2
3. The test methods
includedin this2 Test Guideline2 may
differ in relation to the procedure used to sd02 sd2yi   sd02 measured but sd
y i be used indiscriminately
generate the
data
and
the
readouts
canto address countries’ / 
 / ny  1 
 / n 0  1  

.
=
i





requirements nfor
test
on the Key Event on
activation of dendritic cells of the AOP for skinn yiresults
 0  n 0 
 n yi 
sensitisation while benefiting from the Mutual Acceptance of Data. 

Inh-GAPLA(II-SLR-LA)
Backgroundand principles of the test methods included in the Key Event based
The
Inh-GAPLA
Test Guideline is a ratio of the averaged GAPLA (SLR-LA) for the repetition of the i-th
concentration compared with that with solvent control, and this is written by
4. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classicalmethods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andInh-GAPLA i = 1 / 4  jGAPLA ij / 1 / 4  jGAPLA 0 j .
the Buehler Test (TG406) (4), assess both the induction and elicitation phases of skin sensitisation. The
murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)
Since
GAPLA
is the denominatorof thevalueexclusively,
causes largeandvariationin(5)and theLLNA:
BrdU-ELISA(TG 442 B)(6),nIL8LA,
all assessantheextremely
inductionsmall
response
have also
the nIL8LA.
Therefore,
valueswith an extremely
small valueof Inh-GAPLA
thanwelfare
0.05)
gainedacceptance,since Ind-IL8LA
they provide
an advantage
over the guinea
pig tests
in terms of(lessanimalmight beconsidered
poormeasurement
precision. of the induction phase of skin sensitisation.
togetherwith
an objective











5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; ARENrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals.
6. Test methods described in this TG either quantify the change in the expression of cell surface marker(s)associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skin
sensitisers have been reported to induce the expression of cell membrane markers such as CD40, CD54,
CD80, CD83, and CD86 in addition to induction of proinflammatory cytokines, such as IL-1β and TNF-α,and several chemokines including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
activation (2).
7. However, as DC activation represents only one key event of the skin sensitisation AOP (2) (13),information generated with test methods measuring markers of DC activation alone may not be sufficient
to conclude on the presence or absence of skin sensitisation potential of chemicals. Therefore data
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as nontesting methods, including read-across from chemical analogues (13). Examples of the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

OECD2018
2018©©OECD

62

OECD/OCDE

442E │ 63

o the procedure used to
y to address countries’
APPENDIX V
ls of the AOP for skiny Event based

THE SCHEME OF THE METHODS TO DISSOLVE CHEMICALS FOR THE IL-8 LUC
ASSAY.
(a) For chemicals dissolved in X-VIVOTM 15 at 20 mg/mL

ory animals. The classicalTM
nusson and Kligman, andIf the chemical is soluble in X-VIVO 15
a dilution factor of 5
(1st run )of skin sensitisation. The
Stock solution
20 mg/mL
LLNA: DA (TG 442 A)(4 mg/mL)a dilution factor of 2
xclusively, and have also
terms of animal welfaren.

ssing the first key event 0(OECD TG 442D; AREed for contributing to the

1 mg/mL
4 mg/mL
2 mg/mL
Add to cell suspension in a 96 well plate (50 �L : 50 �L)
0.5 mg/mL
2 mg/mL
1 mg/mL

0

Inh-GAPLAn of cell surface marker(s)posure to sensitisers (e.g.
Determine the highest concentration of the following experiments
e activation of DC. Skiners such as CD40, CD54,
such as IL-1β and TNF-α,
CV05; the lowest concentration at which Inh12), associated with DC
GAPLA becomes <0.05 (the concentration ofstock solution x 1/dilution factor (X) )nsitisation AOP (2) (13),one may not be sufficientemicals. Therefore data
Final concentration in 2nd, 3rd and 4th experimentsupport the discrimination
1 mg/mL 4 mg/mL
n used within Integrated
0
0.5 mg/mL 2 mg/mL
mplementary information,
tion AOP as well as nonples of the use of data 2nd, 3rd or 4th run
ardised both in relation toive predictions, have beenx1
X-VIVOTM 15 control
a dilution factor of 1.5
0.05, neither to sub-categorise
authorities implementing
ent decisions. However,methods may be used on

dilute4xCV05 (4 x the concentrationof stock solution x 1/X )Addition to cells in a 96 well plate (50 �L : 50 �L)
2xCV05©©OECD
OECD2018
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264

TM
3.(b)
The
Fortestchemicals
methodsinsoluble
includedin in
X-VIVO
this Test
15
Guideline
at 20 mg/mL
may differ in relation to the procedure used togenerate the data and the readouts measured but can be used indiscriminately to address countries’
requirements for test results on the Key Event on activation of dendritic cells of the AOP for skinsensitisation
while isbenefitingfrom
the Mutual
Acceptance of Data.
TM 15
If the chemical
insoluble
in X-VIVO(1st run)

Rotate and20 mg/mL centrifuge
Supernatant
x1 (Stock solution)
Background and principles
of
the
test
methods
includedintheKey
Event basedA�dilu on�factor�of�2

Test Guideline

4. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classicalx1/1024
x1/256x1/16
x1/4
methods
that use guinea-pigs,
thex1/64
Guinea Pig Maximisation
Test (GPMT)x1of Magnusson and Kligman, and
0x1/512
x1/128
x1/32
x1/8
x1/2
the Buehler Test (TG 406) (4), assess both the induction and elicitation
phases of skin sensitisation. The
Add to cell suspension in a 96 well plate (50 �L : 50 �L)murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess the induction response exclusively, and have also
gainedx1/2048
acceptance, x1/512
since they provide
over thetests in terms of animal welfare
x1/128 an advantage
x1/32
x1/8guinea pig
x1/2
Final�dilu on
togetherwith x1/1024
an objective x1/256
measurementx1/64
of the induction0
x1/16phase of skinx1/4 sensitisation.

Inh-GAPLA

5. Recently mechanistically-based in chemico and in vitro test methods addressing the first key event(OECD TG 442C; Direct Peptide Reactivity Assay (7)), and second key event (OECD TG 442D; ARENrf2 Luciferase Test Method (8)) of the skin sensitisation AOP have been adopted for contributing to theCV05; the lowest concentration at which Inhevaluation of the skin sensitisation hazard
potential of chemicals.
GAPLA becomes <0.05 (the concentration ofstock solution x 1/dilution factor (X) )6. Test methods described in this TG either quantify the change in the expression of cell surface marker(s)associated with the process of activation of monocytes and DC following exposure to sensitisers (e.g.
CD54, CD86) or the changes in IL-8 expression, a cytokine associated with the activation of DC. Skinrdsensitisers
have been
toriment
induce the expression of cell membrane markers such as CD40, CD54,
Final concentrationin 2nd, 3reported
x1/2048and CD86 in addition to induction
x1/8
x1/2
CD80, CD83,
of proinflammatory cytokines, such as IL-1β and TNF-α,0
x1/1024
x1/16
x1/4
/1024
and several chemokines 1/X
including IL-8 (CXCL8) and CCL3 (9) (10) (11) (12), associated with DC
activation (2).
2nd, 3rd or 4th run
7. However, as DC activation represents only one key event ofdilutethe skin sensitisation AOP (2) (13),information generated with test methods measuring markers
ofDC
activation
may not be sufficient
4xCV05 (4 alone
x the concentration
x1
TM 15 control
X-VIVO
to conclude on the presence or absence of skin sensitisation potential
chemicals.
of stock ofsolutionx 1/X )Therefore dataa dilution factor of 1.5
generated with the test methods described in this Test Guideline are proposed to support the discrimination
between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant complementary information,
Additioncells inof
a 96
plate
(50 �L : 50 �L)L)e.g. derived from in vitro assays addressing otherkeytoevents
thewellskinsensitisation
AOP as well as non2xCV05
testing methods, including read-across from chemical analogues (13). Examples of
the use of data
generated with these methods within Defined Approaches, i.e. approaches standardised both in relation to
the set of information sources used and in the procedure applied to the data to derive predictions, have been
published (13) and can be employed as useful elements within IATA.
0.05

8. The test methods described in this Test Guideline cannot be used on their own, neither to sub-categoriseskin sensitisers into subcategories 1A and 1B as defined by UN GHS (1), for authorities implementing
these two optional subcategories, nor to predict potency for safety assessment decisions. However,depending on the regulatory framework, positive results generated with these methods may be used on
their own to classify a chemical into UN GHS category 1.

OECD2018
2018©©OECD64

OECD/OCDE

442E │ 65

o the procedureAPPENDIX
used toVIy to address countries’
ls of the AOP for skin
THE SCHEME OF THE METHOD TO DISSOLVE 4-NBB FOR THE POSITIVE CONTROLOF THE IL-8 LUC ASSAY.

y Event based

The positive control : 4-NBB (insoluble in X-VIVOTM 15)

ory animals. The classicalnusson and Kligman, andof skin sensitisation. The
LLNA: DA (TG 442 A)
xclusively, and have also
terms of animal welfaren.
20 mg/mlssing the first key event(OECD TG 442D; AREed for contributing to then of cell surface marker(s)posure to sensitisers (e.g.
e activation of DC. Skiners such as CD40, CD54,
such as IL-1β and TNF-α,12), associated with DC

nsitisation AOP (2) (13),one may not be sufficientemicals. Therefore data
support the discriminationn used within Integrated
mplementary information,
tion AOP as well as nonples of the use of data
ardised both in relation toive predictions, have beena dilution factor of 4
Rotate andcentrifuge Supernatant
x1

x1/4

dilution at factor of 2

X-VIVOTM 15

x1/16

control

X-VIVOTM 15
control

x1/8

x1/4

Addition to cells ina 96 well plate (50 �L : 50 �L)

x1/32

x1/16

x1/8

, neither to sub-categorise
authorities implementing
ent decisions. However,methods may be used on©©OECD
OECD20182018

65